2-Carboxamide Cycloamino Ureas

ABSTRACT

The present invention relates to compounds of formula I 
     
       
         
         
             
             
         
       
     
     and salts thereof, wherein the substituents are as defined in the description, to compositions and use of the compounds in the treatment of diseases ameloriated by inhibition of phosphatidylinositol 3-kinase.

This application claims priority to U.S. Provisional Application Ser.No. 61/270,028 filed 2 Jul. 2009, the contents of which are incorporatedherein by reference in their entirety.

The present invention relates to substituted 2-carboxamide cycloaminoureas, as new phosphatidylinositol (PI) 3-kinase inhibitor compounds,their pharmaceutically acceptable salts, prodrugs thereof and processesfor their production. This invention also relates to compositions ofthese compounds, either alone or in combination with at least oneadditional therapeutic agent, and optionally in combination with apharmaceutically acceptable carrier. This invention still furtherrelates to methods of use of these compounds, either alone or incombination with at least one additional therapeutic agent, in theprophylaxis or treatment of a number of diseases, in particular, thosemediated by one or more of abnormal activity of growth factors, receptortyrosine kinases, protein serine/heroine kinases, G protein coupledreceptors and phospholipid kinases and phosphatases.

Phosphatidylinositol 3-kinases (PI3Ks) comprise a family of lipidkinases that catalyze the transfer of phosphate to the D-3′ position ofinositol lipids to produce phosphoinositol-3-phosphate (PIP),phosphoinositol-3,4-diphosphate (PIP₂) andphosphoinositol-3,4,5-triphosphate (PIP₃) that, in turn, act as secondmessengers in signaling cascades by docking proteins containingpleckstrin-homology, FYVE, Phox and other phospholipid-binding domainsinto a variety of signaling complexes often at the plasma membrane((Vanhaesebroeck et al., Annu. Rev. Biochem 70:535 (2001); Katso et al.,Annu. Rev. Cell Dev. Biol. 17:615 (2001)). Of the two Class 1 PI3Ks,Class 1A PI3Ks are heterodimers composed of a catalytic p110 subunit (α,β, δ isoforms) constitutively associated with a regulatory subunit thatcan be p85α, p55α, p50α, p85β or p55γ. The Class 1B sub-class has onefamily member, a heterodimer composed of a catalytic p110γ subunitassociated with one of two regulatory subunits, p101 or p84 (Fruman etal., Annu Rev. Biochem. 67:481 (1998); Suire et al., Curr. Biol. 15:566(2005)). The modular domains of the p85/55/50 subunits include SrcHomology (SH2) domains that bind phosphotyrosine residues in a specificsequence context on activated receptor and cytoplasmic tyrosine kinases,resulting in activation and localization of Class 1A PI3Ks. Class 1BPI3K is activated directly by G protein-coupled receptors that bind adiverse repertoire of peptide and non-peptide ligands (Stephens et al.,Cell 89:105 (1997)); Katso et al., Annu. Rev. Cell Dev. Biol. 17:615-675(2001)). Consequently, the resultant phospholipid products of class IPI3K link upstream receptors with downstream cellular activitiesincluding proliferation, survival, chemotaxis, cellular trafficking,motility, metabolism, inflammatory and allergic responses, transcriptionand translation (Cantley et al., Cell 64:281 (1991); Escobedo andWilliams, Nature 335:85 (1988); Fantl et al., Cell 69:413 (1992)).

In many cases, PIP2 and PIP3 recruit Akt, the product of the humanhomologue of the viral oncogene v-Akt, to the plasma membrane where itacts as a nodal point for many intracellular signaling pathwaysimportant for growth and survival (Fantl et al., Cell 69:413-423(1992);Bader et al., Nature Rev. Cancer 5:921 (2005); Vivanco and Sawyer,Nature Rev. Cancer 2:489 (2002)). Aberrant regulation of PI3K, whichoften increases survival through Akt activation, is one of the mostprevalent events in human cancer and has been shown to occur at multiplelevels. The tumor suppressor gene PTEN, which dephosphorylatesphosphoinositides at the 3′ position of the inositol ring and in sodoing antagonizes PI3K activity, is functionally deleted in a variety oftumors. In other tumors, the genes for the p110α isoform, PIK3CA, andfor Akt are amplified and increased protein expression of their geneproducts has been demonstrated in several human cancers. Furthermore,mutations and translocation of p85α that serve to up-regulate thep85-p110 complex have been described in human cancers. Finally, somaticmissense mutations in PIK3CA that activate downstream signaling pathwayshave been described at significant frequencies in a wide diversity ofhuman cancers (Kang at el., Proc. Natl. Acad. Sci. USA 102:802 (2005);Samuels et al., Science 304:554 (2004); Samuels et al., Cancer Cell7:561-573 (2005)). These observations show that deregulation ofphosphoinositol-3 kinase and the upstream and downstream components ofthis signaling pathway is one of the most common deregulationsassociated with human cancers and proliferative diseases (Parsons etal., Nature 436:792 (2005); Hennessey at el., Nature Rev. Drug Disc.4:988-1004 (2005)).

In view of the above, inhibitors of PI3Ks would be of particular valuein the treatment of proliferative disease and other disorders.Selectivity towards the PI3K α isoform is desirable, and furtherdesirable properties include improved pharmacokinetic properties and/orchemical stability.

WO2004/096797 discloses certain thiazole derivatives as inhibitors ofPI3 kinase and their use as pharmaceutical.

WO 2005/021519 also discloses certain thiazole derivatives as inhibitorsof PI3 kinase and their use as pharmaceutical.

It has now been found that the 2-carboxamide cycloamino ureas of theformula I given below have advantageous pharmacological properties andinhibit, for example, the PI3 kinases (phosphatidylinositol 3-kinase).In particular, preferably, these compounds show selectivity for PI3Kalpha versus beta and/or delta and/or gamma subtypes in the biochemicaland/or in the cellular assay. A further property which is preferablydesirable for compounds of formula I includes improved stability, forexample, improved chemical stability e.g. in solid form and/or in buffersolution. Hence, the compounds of formula I are suitable, for example,to be used in the treatment of diseases depending on the PI3 kinase (inparticular PI3K alpha, such as those showing somatic mutation of PIK3CAor germline mutations or somatic mutation of PTEN), especiallyproliferative diseases such as tumor diseases and leukaemias.

In a first aspect, the present invention provides compounds of formulaI,

wherein,

-   A an unsubstituted or substituted aryl ring or unsubstituted or    substituted heterocyclic ring fused to the rest of the molecule at    the positions indicated by the symbol *;-   X—Y is (CH₂)_(r) or O(CH₂)_(t) or (CH₂)_(t)O wherein,    -   r is 1, 2 or 3;    -   t is 1 or 2;-   n is 0, 1 or 2;-   q is 0, 1, 2, 3 or 4;-   R¹ represents, independently at each occurrence,    -   halo;    -   hydroxy;    -   unsubstituted or substituted aryl;    -   unsubstituted or substituted amino;    -   unsubstituted C₁-C₇-alkyl;    -   C₁-C₇-alkyl, which is substituted one or more times by hydroxy,        C₁-C₇-alkoxy, unsubstituted or substituted amino, aryl or        heterocyclyl, and wherein aryl may be mono or poly-substituted        by halo; or-   two R¹ substituents together form an alkandiyl to form a cyclic    moiety, optionally substituted by hydroxy or halo;-   or a salt, solvate, hydrate or prodrug thereof.

The invention may be more fully appreciated by reference to thefollowing description, including the following glossary of terms and theconcluding examples. As used herein, the terms “including”, “containing”and “comprising” are used herein in their open, non-limiting sense.

Any formula given herein is intended to represent compounds havingstructures depicted by the structural formula as well as certainvariations or forms. In particular, compounds of any formula givenherein may have asymmetric centers and therefore exist in differentstereoisomeric forms such as different enantiomeric forms. If at leastone asymmetrical carbon atom is present in a compound of the formula I,such a compound may exist in optically active form or in the form of amixture of optical isomers, e. g. in the form of a racemic mixture. Thusan asymmetric carbon atom may be present in the (R)-, (S)- or(R,S)-configuration, preferably in the (R)- or (S)-configuration. Alloptical isomers and their mixtures, including the racemic mixtures, arepart of the present invention. Thus, any given formula given herein isintended to represent a racemate, one or more enantiomeric forms, one ormore diastereomeric forms, one or more atropisomeric forms, and mixturesthereof.

Furthermore, certain structures may exist as geometric isomers (e.g. cisand trans isomers), as tautomers, or as atropisomers. For example,substituents at a double bond or a ring may be present in cis-(=Z-) ortrans(=E-) form. The compounds of the invention may thus be present asmixtures of isomers or preferably as pure isomers, preferably asenantiomer-pure diastereomers or pure enantiomers.

Any formula given herein is intended to represent hydrates, solvates,and polymorphs of such compounds, and mixtures thereof.

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine,such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ³¹P, ³²P, ¹⁸F, ³⁵S, ³⁶Cl, ¹²⁵Irespectively. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as ³H,¹³C, and ¹⁴C are incorporated. Such isotopically labelled compounds areuseful in metabolic studies (preferably with ¹⁴C), reaction kineticstudies (with, for example ²H or ³H), detection or imaging techniques,such as positron emission tomography (PET) or single-photon emissioncomputed tomography (SPECT) including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Inparticular, an ¹⁸F or labeled compound may be particularly preferred forPET or SPECT studies. Further, substitution with heavier isotopes suchas deuterium (i.e., ²H) may afford certain therapeutic advantagesresulting from greater metabolic stability, for example increased invivo half-life or reduced dosage requirements. Isotopically labeledcompounds of this invention and prodrugs thereof can generally beprepared by carrying out the procedures disclosed in the schemes or inthe examples and preparations described below by substituting a readilyavailable isotopically labeled reagent for a non-isotopically labeledreagent.

Further, substitution with heavier isotopes, particularly deuterium(i.e., ²H or D) may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example increased in vivo half-life orreduced dosage requirements or an improvement in therapeutic index. Itis understood that deuterium in this context is regarded as asubstituent in the compound of the formula (I). The concentration ofsuch a heavier isotope, specifically deuterium, may be defined by theisotopic enrichment factor. The term “isotopic enrichment factor” asused herein means the ratio between the isotopic abundance and thenatural abundance of a specified isotope. If a substituent in a compoundof this invention is denoted deuterium, such compound has an isotopicenrichment factor for each designated deuterium atom of at least 3500(52.5% deuterium incorporation at each designated deuterium atom), atleast 4000 (60% deuterium incorporation), at least 4500 (67.5% deuteriumincorporation), at least 5000 (75% deuterium incorporation), at least5500 (82.5% deuterium incorporation), at least 6000 (90% deuteriumincorporation), at least 6333.3 (95% deuterium incorporation), at least6466.7 (97% deuterium incorporation), at least 6600 (99% deuteriumincorporation), or at least 6633.3 (99.5% deuterium incorporation). Inthe compounds of this invention any atom not specifically designated asa particular isotope is meant to represent any stable isotope of thatatom. Unless otherwise stated, when a position is designatedspecifically as “H” or “hydrogen”, the position is understood to havehydrogen at its natural abundance isotopic composition. Accordingly, inthe compounds of this invention any atom specifically designated as adeuterium (D) is meant to represent deuterium, for example in the rangesgiven above.

When referring to any formula given herein, the selection of aparticular moiety from a list of possible species for a specifiedvariable is not intended to define the moiety for the variable appearingelsewhere. In other words, where a variable appears more than once, thechoice of the species from a specified list is independent of the choiceof the species for the same variable elsewhere in the formula (where oneor more up to all more general expressions in embodiments characterizedas preferred above or below can be replaced with a more specificdefinition, thus leading to a more preferred embodiment of theinvention, respectively).

Where the plural form (e.g. compounds, salts, pharmaceuticalpreparations, diseases and the like) is used, this includes the singular(e.g. a single compound, a single salt, a single pharmaceuticalpreparation, a single disease, and the like). “A compound” does notexclude that (e.g. in a pharmaceutical formulation) more than onecompound of the formula (I) (or a salt thereof) is present.

Salts are preferably the pharmaceutically acceptable salts of compoundsof formula (I) if they are carrying salt-forming groups. Acids/basesrequired to form the salts are generally known in the field.

The following general definitions shall apply in this specification,unless otherwise specified:

Halogen (or halo) denotes fluorine, bromine, chlorine or iodine, inparticular fluorine, chlorine. Halogen-substituted groups and moieties,such as alkyl substituted by halogen (halogenalkyl) can be mono-, poly-or per-halogenated.

Hetero atoms are atoms other than Carbon and Hydrogen, preferablynitrogen (N), oxygen (O) or sulfur (S), in particular nitrogen.

“Alkyl” refers to a straight-chain or branched-chain alkyl group, andincludes C₁₋₇alkyl and more preferably C₁₋₄alkyl. Such alkyl groupsinclude, for example, methyl, ethyl, n- or iso-propyl, n-, iso-, sec- ortert-butyl, n-pentyl, n-hexyl, n-heptyl, with particular preferencegiven to methyl, ethyl, n-propyl, iso-propyl, n-butyl and iso-butyl.Alkyl may be unsubstituted or substituted. Exemplary substituentsinclude, but are not limited to hydroxy, alkoxy, halogen (especiallyfluoro), amino and di-substituted amino, mono- or di-alkyl substitutedamino, acetylamino, morpholinyl, aryl. An example of a substituted alkylis trifluoromethyl. Cycloalkyl may also be a substituent to alkyl. Anexample of such a case is the moiety (alkyl)-cycloalkyl, such as(alkyl)-cyclopropyl or (alkyl)-cyclobutyl, e.g. methyl-cyclopropyl ormethyl-cyclobutyl. A more specific example of an (alkyl)-cycloalkylmoiety includes geminal-type of substitution pattern, e.g. 1-alkylcycloalkyl, such as 1-methyl cyclopropyl. Another example of cycloalkylas a substituent to alkyl is alkandiyl-cycloalkyl, such asalkandiyl-cyclopropyl, e.g. —CH₂-cyclopropyl. C₁-C₇-alkyl is alkyl withfrom and including 1 up to and including 7 carbon atoms, preferably fromand including 1 up to and including 4 carbon atoms (C₁-C₄-alkyl), and islinear or branched; preferably, lower alkyl is butyl, such as n-butyl,sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or isopropyl,ethyl or preferably methyl.

Each alkyl part of other groups like “alkoxy”, “alkoxyalkyl”,“alkoxycarbonyl”, “alkoxy-carbonylalkyl”, “alkylsulfonyl”,“alkylsulfoxyl”, “alkylamino”, “halogenalkyl” shall have the samemeaning as described in the above-mentioned definition of “alkyl”.

“C₃₋₇-Cycloalkyl” refers to a saturated or partially saturated,monocyclic, fused polycyclic, or Spiro polycyclic, carbocycle havingfrom 3 to 7 ring atoms per carbocycle. Illustrative examples ofcycloalkyl groups include the following moieties: cyclopropyl,cyclobutyl, cyclpentyl and cylclohexyl. C₃-C₇-cycloalkyl may beunsubstituted or substituted; exemplary substituents are provided in thedefinition for alkyl. C₃-C₇-cycloalkyl may also be a substituent onother groups, e.g. on an alkyl group.

“Aryl” refers to an unsaturated carbocyclic aromatic ring system,preferably, having a ring system of not more than 16 carbon atoms,especially not more than 10 carbon atoms, e.g. having 6 to 16,preferably 6 to 10 ring carbon atoms, is preferably mono- or bi-cyclic,and is unsubstituted or substituted. For example, aryl is unsubstitutedor substituted phenyl.

“Heterocyclyl” refers to a heterocyclic radical that is unsaturated (inparticular maximally unsaturated, eg. carrying the highest possiblenumber of conjugated double bonds in the ring(s)) e.g. heteroaryl),saturated or partially saturated in the bonding ring and is preferably amonocyclic or in a broader aspect of the invention bicyclic ring; has3-16 ring atoms, more preferably 4-10 ring atoms, wherein at least inthe ring bonding to the radical of the molecule of formula (I) one ormore, preferably 1-4 ring atoms, especially one or two ring atoms are aheteroatom selected from the group consisting of nitrogen, oxygen andsulfur; the bonding ring preferably having 4-12 ring atoms, especially4-7 ring atoms, for example 6-10 ring atoms, especially for heteroaryl,such as 5, 6, 9 or 10 ring atoms. The heterocyclyl may be unsubstitutedor substituted by one or more, especially 1 to 3, substituentsindependently selected from the group consisting of alkyl or thesubstituents defined above for substituted alkyl and/or from one or moreof the following substituents: oxo (═O), thiocarbonyl (═S), imino (═NH),imino-lower alkyl, and, for nitrogen containing heteroaryls, includingN-oxides thereof.

“Treatment” includes prophylactic (preventive) and therapeutic treatmentas well as the delay of progression of a disease or disorder.

“PI3 kinase mediated diseases” (especially PI3K alpha mediated diseases)are especially such disorders that respond in a beneficial way (e.g.amelioration of one or more symptoms, delay of the onset of a disease,up to temporary or complete cure from a disease) to the inhibition of aPI3 kinase, especially inhibition of PI3Kalpha (where the diseases to betreated may include those showing somatic mutation of PIK3CA or germlinemutations or somatic mutation of PTEN). Diseases to be treated includeespecially proliferative diseases such as tumor diseases, includingsolid tumors, leukaemias, glioblastoma, breast cancer and prostatecancer may be mentioned).

“Salts” (which, what is meant by “or salts thereof” or “or a saltthereof”), can be present alone or in mixture with free compound of theformula I and are preferably pharmaceutically acceptable salts.Salt-forming groups in a compound of formula (I) are groups or radicalshaving basic or acidic properties. Compounds having at least one basicgroup or at least one basic radical, e.g., amino; a secondary aminogroup not forming a peptide bond or a pyridyl radical, may form acidaddition salts, e.g., with inorganic acids, such as hydrochloric acid,sulfuric acid or a phosphoric acid; or with suitable organic carboxylicor sulfonic acids, e.g., aliphatic mono- or di-carboxylic acids, such astrifluoroacetic acid, acetic acid, propionic acid, glycolic acid,succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malicacid, tartaric acid, citric acid or oxalic acid; or amino acids, such asarginine or lysine; aromatic carboxylic acids, such as benzoic acid;2-phenoxy-benzoic acid; 2-acetoxy-benzoic acid; salicylic acid;4-aminosalicylic acid; aromatic-aliphatic carboxylic acids, such asmandelic acid or cinnamic acid; heteroaromatic carboxylic acids, such asnicotinic acid or isonicotinic acid; aliphatic sulfonic acids, such asmethane-, ethane- or 2-hydroxyethanesulfonic acid; or aromatic sulfonicacids, e.g., benzene-, p-toluene- or naphthalene-2-sulfonic acid. Whenseveral basic groups are present mono- or poly-acid addition salts maybe formed.

Compounds of formula (I) having acidic groups, a carboxy group or aphenolic hydroxy group, may form metal or ammonium salts, such as alkalimetal or alkaline earth metal salts, e.g., sodium, potassium, magnesiumor calcium salts; or ammonium salts with ammonia or suitable organicamines, such as tertiary monoamines, e.g., triethylamine ortri(2-hydroxyethyl)-amine, or heterocyclic bases, e.g.,N-ethyl-piperidine or N,N′-dimethylpiperazine. Mixtures of salts arepossible.

Compounds of formula (I) having both acidic and basic groups can forminternal salts.

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. For therapeutic use, only pharmaceutically acceptablesalts or free compounds are employed (where applicable in the form ofpharmaceutical preparations), and these are therefore preferred. In viewof the close relationship between the novel compounds in free form andthose in the form of their salts, including those salts that can be usedas intermediates, for example in the purification or identification ofthe novel compounds, any reference to the free compounds hereinbeforeand hereinafter is to be understood as referring also to thecorresponding salts, as appropriate and expedient.

Compounds of the present invention may also form solvates and hydrates,and as such any reference to a compound of formula (I) is therefore tobe understood as referring also to the corresponding solvate and/orhydrate of the compound of formula (I), as appropriate and expedient.

The present invention also relates to pro-drugs of a compound of formula(I) that convert in vivo to the compound of formula (I) as such. Anyreference to a compound of formula (I) is therefore to be understood asreferring also to the corresponding pro-drugs of the compound of formula(I), as appropriate and expedient.

Combination refers to either a fixed combination in one dosage unitform, or a kit of parts for the combined administration where a compoundof the formula I and a combination partner (e.g. an other drug asexplained below, also referred to as “therapeutic agent” or “co-agent”)may be administered independently at the same time or separately withintime intervals, especially where these time intervals allow that thecombination partners show a cooperative, e.g. synergistic effect. Theterms “co-administration” or “combined administration” or the like asutilized herein are meant to encompass administration of the selectedcombination partner to a single subject in need thereof (e.g. apatient), and are intended to include treatment regimens in which theagents are not necessarily administered by the same route ofadministration or at the same time. The term “pharmaceuticalcombination” as used herein means a product that results from the mixingor combining of more than one active ingredient and includes both fixedand non-fixed combinations of the active ingredients. The term “fixedcombination” means that the active ingredients, e.g. a compound offormula I and a combination partner, are both administered to a patientsimultaneously in the form of a single entity or dosage. The term“non-fixed combination” means that the active ingredients, e.g. acompound of formula I and a combination partner, are both administeredto a patient as separate entities either simultaneously, concurrently orsequentially with no specific time limits, wherein such administrationprovides therapeutically effective levels of the two compounds in thebody of the patient. The latter also applies to cocktail therapy, e.g.the administration of three or more active ingredients.

In preferred embodiments, which are preferred independently,collectively or in any combination or sub-combination, the inventionrelates to a compound of the formula I, in free base form or in saltform, wherein the substituents are as defined herein.

As shown in formula I, the alpha-amide substituent is at the 2-positionon the pyrrolidine ring and the stereochemistry at this position is asdrawn.

The ring A is preferably an unsubstituted or substituted 5- or6-membered heterocyclic (preferably a heteroaryl) ring containing 1 or 2heteroatoms selected from N, S or O, wherein at least one heteroatom isN.

More preferably, ring A is selected from an unsubstituted or substitutedpyridine ring, unsubstituted or substituted pyrimidine ring,unsubstituted or substituted thiazole ring, unsubstituted or substitutedpyrazole ring or unsubstituted or substituted oxazole ring. Morepreferred is an unsubstituted or substituted pyridine ring,unsubstituted or substituted pyrimidine ring or unsubstituted orsubstituted thiazole ring.

Preferably, ring A is fused to the rest of the molecule of formula Ithrough carbon atoms of ring A.

Ring A is preferably substituted by one, two or three R² groups,preferably two R² groups, most preferably one R² group, independentlyselected at each occurrence from,

-   -   unsubstituted or substituted C₁-C₇-alkyl;    -   unsubstituted or substituted amino;    -   unsubstituted or substituted C₃-C₇-cycloalkyl.

Preferably, R² is selected from

-   -   unsubstituted C₁-C₇-alkyl;    -   di(C₁-C7alkyl)amino;    -   C₁-C₇-alkyl substituted one or more times by C₃-C₇-cycloalkyl or        halo (preferably fluoro);    -   unsubstituted C₃-C₇-cycloalkyl;    -   C₃-C₇-cycloalkyl which is substituted one or more times by halo        (preferably fluoro), (halo-C₁-C₇-alkyl) or C₁-C₇-alkyl;

More preferably, R² is selected from methyl, t-butyl, diethylamino,cyclopropylmethyl, 2-fluoro-1,1-dimethylethyl or2,2,2-trifluoro-1,1-dimethyl-ethyl.

In another embodiment, R² is selected from methyl, t-butyl,diethylamino, cyclopropylmethyl or 2-fluoro-1,1-dimethylethyl.

The ring A is more preferably selected from A1 or A2 or A3 or A4 or A5or A6:

wherein,

Z is N or CH and R² is defined as above.

Preferably, ring A is selected from A1 or A2.

X—Y preferably represents (CH₂)_(r) or O(CH₂)_(t) wherein,

-   -   r is 1, 2 or 3;    -   t is 1 or 2;

-   X—Y more preferably represents (CH₂)_(r) or O(CH₂)_(t) wherein r is    2 and t is 1. For the avoidance of doubt, that is, X—Y is preferably    —CH₂—CH₂— or —O—CH₂—, such that in the latter case, the X in X—Y is    the O in —O—CH₂—.

R¹ preferably represents, independently at each occurrence,

-   -   halo;    -   hydroxy;    -   unsubstituted or substituted phenyl;    -   di(C₁-C₇-alkyl)amino;    -   unsubstituted C₁-C₇-alkyl;    -   C₁-C₇-alkyl, which is substituted one or more times by hydroxy,        C₁-C₇-alkoxy, di(C₁-C₇-alkyl)amino,        di-(perdeuteroC₁-C₇-alkyl)amino, phenyl, morpholinyl,        acetylamino, or N—(C₁-C₇-alkyl)-N-(phenylC₁-C₇-alkyl)amino, and        wherein independently each phenyl may be mono or        poly-substituted by halo.

R¹ more preferably represents, independently at each occurrence,

-   -   fluoro;    -   hydroxy;    -   unsubstituted phenyl;    -   dimethylamino;    -   methyl;    -   methyl, which is substituted one or more times (preferably        substituted once) by hydroxy, methoxy, dimethylamino,        di-(perdeuteromethyl)amino, phenyl, morpholinyl, acetylamino, or        N-(methyl)-N-(phenylmethyl)amino, and wherein independently each        phenyl may be mono or poly-substituted by fluoro.

R¹ most preferably represents, independently at each occurrence,

-   -   fluoro;    -   hydroxy;    -   unsubstituted phenyl;    -   dimethylamino;    -   methyl;    -   hydroxy methyl;    -   methoxy methyl;    -   dimethylamino methyl;    -   di-(perdeuteromethyl)amino methyl;    -   benzyl;    -   morpholin-4-yl methyl;    -   N-acetylamino methyl;    -   N-(methyl)-N-(3-fluoro-phenylmethyl)amino methyl.

An embodiment of the present invention includes compounds of the formulaI wherein n is 0 or 1. Preferably, n is 1.

Another embodiment of the present invention includes compounds of theformula I wherein q is 0, that is, wherein the nitrogen containingheterocyclic ring is substituted only by the amide at position 2. Inthis embodiment, it is preferred that n is 0 or 1, more preferably 1.

Another embodiment of the present invention includes compounds of theformula I wherein q is 1, that is, wherein the nitrogen containingheterocyclic ring is substituted only by the amide at position 2 and asingle R¹ group. In this embodiment it is preferred that n is 0 or 1,more preferably 1. In this embodiment, the R¹ group may be substitutedat position 2- (i.e. on the same carbon as that which is substituted bythe amide group) or position 3- or position 4- or position 5- of thenitrogen containing heterocyclic ring.

Preferably, in this embodiment, the R¹ group is substituted at position3 of the nitrogen containing heterocyclic ring, i.e. compounds offormula IA:

wherein the substitutents are defined as for a compound of formula (I).

Preferably in compounds of formula IA, n is 1, thus providing compoundswherein the nitrogen containing heterocyclic ring is a pyrrolidine ring,substituted at the 2-position by an amide having the drawnstereochemistry, and in the 3-position by an R¹ group.

Preferably, the R¹ group has a stereochemistry which is cis- to theamide at position 2, i.e. compounds according to formula (IA′):

wherein the substitutents are defined as for a compound of formula (I).

Preferably in compounds of formula IA′, n is 1, thus providing compoundswherein the nitrogen containing heterocyclic ring is a pyrrolidine ring,substituted at the 2-position by an amide having the drawnstereochemistry, and in the 3-position by an R¹ group having the drawnstereochemisty, thus the amide and R¹ groups are cis-relative to eachother.

A further embodiment of the present invention includes compounds of theformula I wherein q is 2 or 3, thus at least two R¹ substituents arepresent, each R¹ independently selected from the groups defined as forformula I herein. In this embodiment, it is preferred that at least eachof two R¹ is bonded at position 3 of the pyrrolidine ring, and anoptional third R¹ group, if present, is bonded elsewhere on the nitrogencontaining heterocyclic ring. It is further preferred that n is 1 andthe third R¹ group, if present, is bonded at the 4- or 5-position of theresultant pyrrolidine ring, i.e. to provide compounds of formula IB:

wherein the substituents are defined as for a compound of formula (I).

In compounds according to formula IB, it is preferred that the third R¹is bonded at position 4- of the pyrrolidine ring.

A further embodiment of the present invention includes compounds of theformula I wherein n is 1, and wherein two R¹ groups are bonded atposition 3 of the pyrrolidine ring, and, together form an alkandiyl,preferably a C₃-C₈-cycloalkyl, in particular a cyclopropyl, i.e. toprovide compounds of formula IC:

wherein the substitutents are defined as for a compound of formula (I)and the third R¹ group is optional, and if present, is preferably bondedat position 4- of the pyrrolidine ring.

In any of the formulae (IA), (IA′), (IB) or (IC), the preferreddefinitions, if not otherwise stated, for ring A, X—Y, R¹ and n can alsoapply.

The invention further relates to pharmaceutically acceptable prodrugs ofa compound of formula (I), (IA), (IA′), (IB) and/or (IC).

The invention further relates to pharmaceutically acceptable metabolitesof a compound of formula (I), (IA), (IA′), (IB) and/or (IC).

The invention relates especially to the compounds of the formula (I),(IA), (IA′), (IB) and/or (IC) given in the Examples, as well as themethods of manufacture described herein.

The present invention also relates to processes for the production of acompound of formula (I), (IA), (IA′), (IB) and/or (IC). In principle allknown processes which convert two different amines into a correspondingurea derivative are suitable and may be applied by using the respectivestarting material.

Thus, the invention in particular relates to a process which comprisesreacting a compound of formula II

wherein the substituents are as defined above, either with a compound offormula IIIA

wherein the substituents are as defined above, in the presence of anactivating agent (“method A”) or with a compound of formula IIIB

wherein R¹ is as defined above; RG represents a reactive group (such asimidazolylcarbonyl) (“method B”),

in each case optionally in the presence of a diluent and optionally inthe presence of a reaction aid and

recovering the resulting compound of formula I in free form or in formof a salt and, optionally converting a compound of the formula Iobtainable according to method A or method B into a different compoundof the formula I, and/or converting an obtainable salt of a compound ofthe formula I into a different salt thereof, and/or converting anobtainable free compound of the formula I into a salt thereof, and/orseparating an obtainable isomer of a compound of the formula I from oneor more different obtainable isomers of the formula I.

Reaction Conditions

The process may be performed according to methods known in the art, oras disclosed below in the Examples. For example a compound of formula IImay be reacted with a compound of formula IIIA or IIIB in a solvent,e.g. dimethylformamide, in the presence of a base e.g. an organic amine,e.g. triethylamine.

Where temperatures are given hereinbefore or hereinafter, “about” has tobe added, as minor deviations from the numeric values given, e.g.variations of ±10%, are typically tolerable. All reactions may takeplace in the presence of one or more diluents and/or solvents. Thestarting materials may be used in equimolar amounts; alternatively, acompound may be used in excess, e.g. to function as a solvent or toshift equilibrium or to generally accelerate reaction rates.

Reaction aids, such as acids, bases or catalysts may be added insuitable amounts, as known in the field, required by a reaction and inline with generally known procedures.

Protecting Groups

If one or more other functional groups, for example carboxy, hydroxy,amino, sulfhydryl or the like are or need to be protected in a startingmaterial as described herein or any other precursor, because they shouldnot take part in the reaction or disturb the reaction, these are suchgroups as are usually used in the synthesis of peptide compounds, andalso of cephalosporins and penicillins, as well as nucleic acidderivatives and sugars. Protecting groups are such groups that are nolonger present in the final compounds once they are removed, whilegroups that remain as substituents are not protecting groups in thesense used here which are groups that are added at a starting materialor intermediate stage and removed to obtain a final compound. Also inthe case of conversions of a compound of the formula (I), (IA), (IA′),(IB) and/or (IC) into a different compound of the formula (I), (IA),(IA′), (IB) and/or (IC), protecting groups may be introduced andremoved, if useful or required. The protecting groups may already bepresent in precursors and should protect the functional groups concernedagainst unwanted secondary reactions, such as acylations,etherifications, esterifications, oxidations, solvolysis, and similarreactions. It is a characteristic of protecting groups that they lendthemselves readily, i.e. without undesired secondary reactions, toremoval, typically by acetolysis, protonolysis, solvolysis, reduction,photolysis or also by enzyme activity, for example under conditionsanalogous to physiological conditions, and that they are not present inthe end-products. The specialist knows, or can easily establish, whichprotecting groups are suitable with the reactions mentioned above andbelow.

The protection of such functional groups by such protecting groups, theprotecting groups themselves, and their removal reactions are describedfor example in standard reference works, such as J. F. W. McOmie,“Protective Groups in Organic Chemistry”, Plenum Press, London and NewYork 1973, in T. W. Greene, “Protective Groups in Organic Synthesis”,Third edition, Wiley, New York 1999, in “The Peptides”; Volume 3(editors: E. Gross and J. Meienhofer), Academic Press, London and NewYork 1981, in “Methoden der organischen Chemie” (Methods of organicchemistry), Houben Weyl, 4th edition, Volume 15/l, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosäuren, Peptide,Proteine” (Amino acids, peptides, proteins), Verlag Chemie, Weinheim,Deerfield Beach, and Basel 1982, and in Jochen Lehmann, “Chemie derKohlenhydrate: Monosaccharide and Derivate” (Chemistry of carbohydrates:monosaccharides and derivatives), Georg Thieme Verlag, Stuttgart 1974.

Optional Reactions and Conversions

A compound of the formula (I), (IA), (IA′), (IB) and/or (IC) may beconverted into a different compound of the formula (I), (IA), (IA′),(IB) and/or (IC).

In a compound of the formula (I), (IA), (IA′), (IB) and/or (IC) whereina substituent carries an amino or amino-C₁-C₇-alkyl substituent, theamino can be converted into acylamino, e.g. C₁-C₇-alkanoylamino, byreaction with a corresponding C₁-C₇-alkanoylhalogenide, e.g. acorresponding chloride, in the presence of a tertiary nitrogen base,such as triethylamine or pyridine, in the absence or presence of anappropriate solvent, such a methylene chloride, for example attemperatures in the range from −20 to 50° C., e.g. at about roomtemperature.

Salts of a compound of formula (I), (IA), (IA′), (IB) and/or (IC) with asalt-forming group may be prepared in a manner known per se. Acidaddition salts of compounds of formula (I), (IA), (IA′), (IB) and/or(IC) may thus be obtained by treatment with an acid or with a suitableanion exchange reagent. A salt with two acid molecules (for example adihalogenide of a compound of formula (I), (IA), (IA′), (IB) and/or(IC)) may also be converted into a salt with one acid molecule percompound (for example a monohalogenide); this may be done by heating toa melt, or for example by heating as a solid under a high vacuum atelevated temperature, for example from 130 to 170° C., one molecule ofthe acid being expelled per molecule of a compound of formula (I), (IA),(IA′), (IB) and/or (IC). Salts can usually be converted to freecompounds, e.g. by treating with suitable basic compounds, for examplewith alkali metal carbonates, alkali metal hydrogencarbonates, or alkalimetal hydroxides, typically potassium carbonate or sodium hydroxide.

Stereoisomeric mixtures, e.g. mixtures of diastereomers, can beseparated into their corresponding isomers in a manner known per se bymeans of suitable separation methods. Diastereomeric mixtures forexample may be separated into their individual diastereomers by means offractionated crystallization, chromatography, solvent distribution, andsimilar procedures. This separation may take place either at the levelof a starting compound or in a compound of formula (I), (IA), (IA′),(IB) and/or (IC) itself. Enantiomers may be separated through theformation of diastereomeric salts, for example by salt formation with anenantiomer-pure chiral acid, or by means of chromatography, for exampleby HPLC, using chromatographic substrates with chiral ligands.

It should be emphasized that reactions analogous to the conversionsmentioned herein may also take place at the level of appropriateintermediates (and are thus useful in the preparation of correspondingstarting materials).

Starting Materials:

The starting materials of the formulae II and III, as well as otherstarting materials mentioned herein, e.g. below, can be preparedaccording to or in analogy to methods that are known in the art, areknown in the art and/or are commercially available. Insofar as theproduction of the starting materials is not particularly described, thecompounds are either known or may be prepared analogously to methodsknown in the art, e.g. in WO 05/021519 or WO04/096797, or as disclosedhereinafter. Novel starting materials, as well as processes for thepreparation thereof, are likewise an embodiment of the presentinvention. In the preferred embodiments, such starting materials areused and the reaction chosen are selected so as to enable the preferredcompounds to be obtained.

In the starting materials (including intermediates), which may also beused and/or obtained as salts where appropriate and expedient, thesubstituents are preferably as defined for a compound of the formula(I), (IA), (IA′), (IB) and/or (IC).

-   -   Pharmaceutical compositions, uses and methods of treatment    -   The present invention also relates to use of the compounds of        formula (I), (IA), (IA′), (IB) and/or (IC) as disclosed herein        as pharmaceuticals. The present invention includes in one        embodiment compositions comprising a compound of formula (I),        (IA), (IA′), (IB) and/or (IC), e.g. for human or veterinary use,        e.g. where inhibition of PI3K is indicated.    -   In one embodiment, the invention relates to the treatment of        cellular proliferative diseases such as tumor (benign or        malignant) and/or cancerous cell growth, e.g. mediated by PI3K.        Diseases may include those showing somatic mutation of PIK3CA or        germline mutations or somatic mutation of PTEN. In particular,        the compounds may be useful in the treatment of human or animal        (e.g., murine) cancers, including, for example, sarcoma; lung;        bronchus; prostate; breast (including sporadic breast cancers        and sufferers of Cowden disease); pancreas; gastrointestinal        cancer; colon; rectum; colon carcinoma; colorectal adenoma;        thyroid; liver; intrahepatic bile duct; hepatocellular; adrenal        gland; stomach; gastric; glioma; glioblastoma; endometrial;        melanoma; kidney; renal pelvis; urinary bladder; uterine corpus;        uterine cervix; vagina; ovary; multiple myeloma; esophagus; a        leukaemia; acute myelogenous leukemia; chronic myelogenous        leukemia; lymphocytic leukemia; myeloid leukemia; brain; a        carcinoma of the brain; oral cavity and pharynx; larynx; small        intestine; non-Hodgkin lymphoma; melanoma; villous colon        adenoma; a neoplasia; a neoplasia of epithelial character;        lymphomas; a mammary carcinoma; basal cell carcinoma; squamous        cell carcinoma; actinic keratosis; tumor diseases, including        solid tumors; a tumor of the neck or head; polycythemia Vera;        essential thrombocythemia; and myelofibrosis with myeloid        metaplasia.

In other embodiments, the condition or disorder (e.g. PI3K-mediated) isselected from the group consisting of: an epidermal hyperproliferation,prostate hyperplasia, a neoplasia, a neoplasia of epithelial character,Cowden syndrome, Lhermitte-Dudos disease or Bannayan-Zonana syndrome,asthma, COPD, ARDS, Loffler's syndrome, eosinophilic pneumonia,parasitic (in particular metazoan) infestation (including tropicaleosinophilia), bronchopulmonary aspergillosis, polyarteritis nodosa(including Churg-Strauss syndrome), eosinophilic granuloma,eosinophil-related disorders affecting the airways occasioned bydrug-reaction, psoriasis, contact dermatitis, atopic dermatitis,alopecia areata, erythema multiforme, dermatitis herpetiformis,scleroderma, vitiligo, hypersensitivity angiitis, urticaria, bullouspemphigoid, lupus erythematosus, pemphisus, epidermolysis bullosaacquisita, autoimmune haematogical disorders (e.g. haemolytic anaemia,aplastic anaemia, pure red cell anaemia and idiopathicthrombocytopenia), systemic lupus erythematosus, polychondritis,scleroderma, Wegener granulomatosis, dermatomyositis, chronic activehepatitis, myasthenia gravis, Steven-Johnson syndrome, idiopathic sprue,autoimmune inflammatory bowel disease (e.g. ulcerative colitis andCrohn's disease), endocrine opthalmopathy, Grave's disease, sarcoidosis,alveolitis, chronic hypersensitivity pneumonitis, multiple sclerosis,primary biliary cirrhosis, uveitis (anterior and posterior),interstitial lung fibrosis, psoriatic arthritis, glomerulonephritis,cardiovascular diseases, atherosclerosis, hypertension, deep venousthrombosis, stroke, myocardial infarction, unstable angina,thromboembolism, pulmonary embolism, thrombolytic diseases, acutearterial ischemia, peripheral thrombotic occlusions, and coronary arterydisease, reperfusion injuries, retinopathy, such as diabetic retinopathyor hyperbaric oxygen-induced retinopathy, and conditions characterizedby elevated intraocular pressure or secretion of ocular aqueous humor,such as glaucoma.

For the above uses the required dosage will of course vary depending onthe mode of administration, the particular condition to be treated andthe effect desired. In general, satisfactory results are indicated to beobtained systemically at daily dosages of from about 0.03 to 10.0 mg/kgper body weight. An indicated daily dosage in the larger mammal, e.g.humans, is in the range from about 0.5 mg to about 1 g, convenientlyadministered, for example, in divided doses up to four times a day or inretard form. Suitable unit dosage forms for oral administration comprisefrom ca. 0.1 to 500 mg active ingredient.

The compounds of formula (I), (IA), (IA′), (IB) and/or (IC) may beadministered by any conventional route, in particular enterally, e.g.orally, e.g. in the form of tablets or capsules, or parenterally, e.g.in the form of injectable solutions or suspensions, topically, e.g. inthe form of lotions, gels, ointments or creams, by inhalation,intranasally, or in a suppository form.

The compounds of formula (I), (IA), (IA′), (IB) and/or (IC) may beadministered in free form or in pharmaceutically acceptable salt forme.g. as indicated above. Such salts may be prepared in conventionalmanner and exhibit the same order of activity as the free compounds.

Consequently, the invention also provides:

-   -   a method for preventing or treating conditions, disorders or        diseases mediated by the activation of the PI3, e.g. the PI3        kinase alpha enzyme e.g. such as indicated above, in a subject        in need of such treatment, which method comprises administering        to said subject an effective amount of a compound of formula        (I), (IA), (IA′), (IB) and/or (IC) or a pharmaceutically        acceptable salt thereof    -   use of a compound of formula (I), (IA), (IA′), (IB) and/or (IC),        in free form or in a pharmaceutically acceptable salt form as a        pharmaceutical, e.g. in any of the methods as indicated herein.    -   a compound of the formula (I), (IA), (IA′), (IB) and/or (IC) in        free form or in pharmaceutically acceptable salt form for use as        pharmaceutical, e.g. in any of the methods as indicated herein,        in particular for the use in one or more phosphatidylinositol        3-kinase mediated diseases.    -   the use of a compound of formula (I), (IA), (IA′), (IB) and/or        (IC) in free form or in pharmaceutically acceptable salt form in        any of the methods as indicated herein, in particular for the        treatment of one or more phosphatidylinositol 3-kinase mediated        diseases.    -   the use of a compound of formula (I), (IA), (IA′), (IB) and/or        (IC) in free form or in pharmaceutically acceptable salt form in        any of the methods as indicated herein, in particular for the        manufacture of a medicament for the treatment of one or more        phosphatidylinositol 3-kinase mediated diseases.

PI3K serves as a second messenger node that integrates parallelsignaling pathways, evidence is emerging that the combination of a PI3Kinhibitor with inhibitors of other pathways will be useful in treatingcancer and proliferative diseases in humans. Approximately 20-30% ofhuman breast cancers overexpress Her-2/neu-ErbB2, the target for thedrug trastuzumab. Although trastuzumab has demonstrated durableresponses in some patients expressing Her2/neu-ErbB2, only a subset ofthese patients respond. Recent work has indicated that this limitedresponse rate can be substantially improved by the combination oftrastuzumab with inhibitors of PI3K or the PI3K/AKT pathway (Chan etal., Breast Can. Res. Treat. 91:187 (2005), Woods Ignatoski et al.,Brit. J. Cancer 82:666 (2000), Nagata et al., Cancer Cell 6:117 (2004)).

A variety of human malignancies express activitating mutations orincreased levels of Her1/EGFR and a number of antibody and smallmolecule inhibitors have been developed against this receptor tyrosinekinase including tarceva, gefitinib and erbitux. However, while EGFRinhibitors demonstrate anti-tumor activity in certain human tumors(e.g., NSCLC), they fail to increase overall patient survival in allpatients with EGFR-expressing tumors. This may be rationalized by thefact that many downstream targets of Her1/EGFR are mutated orderegulated at high frequencies in a variety of malignancies, includingthe PI3K/Akt pathway. For example, gefitinib inhibits the growth of anadenocarcinoma cell line in in vitro assays. Nonetheless, sub-clones ofthese cell lines can be selected that are resistant to gefitinib thatdemonstrate increased activation of the PI3/Akt pathway. Down-regulationor inhibition of this pathway renders the resistant sub-clones sensitiveto gefitinib (Kokubo et al., Brit. J. Cancer 92:1711 (2005)).Furthermore, in an in vitro model of breast cancer with a cell line thatharbors a PTEN mutation and over-expresses EGFR inhibition of both thePI3K/Akt pathway and EGFR produced a synergistic effect (She et al.,Cancer Cell 8:287-297(2005)). These results indicate that thecombination of gefitinib and PI3K/Akt pathway inhibitors would be anattractive therapeutic strategy in cancer.

The combination of AEE778 (an inhibitor of Her-2/neu/ErbB2, VEGFR andEGFR) and RAD001 (an inhibitor of mTOR, a downstream target of Akt)produced greater combined efficacy that either agent alone in aglioblastoma xenograft model (Goudar et al., Mol. Cancer. Ther.4:101-112 (2005)).

Anti-estrogens, such as tamoxifen, inhibit breast cancer growth throughinduction of cell cycle arrest that requires the action of the cellcycle inhibitor p27Kip. Recently, it has been shown that activation ofthe Ras-Raf-MAP Kinase pathway alters the phosphorylation status ofp27Kip such that its inhibitory activity in arresting the cell cycle isattenuated, thereby contributing to anti-estrogen resistance (Donovan,et al, J. Biol. Chem. 276:40888, (2001)). As reported by Donovan et al.,inhibition of MAPK signaling through treatment with MEK inhibitorreversed the aberrant phosphorylation status of p27 in hormonerefractory breast cancer cell lines and in so doing restored hormonesensitivity. Similarly, phosphorylation of p27Kip by Akt also abrogatesits role to arrest the cell cycle (Viglietto et al., Nat Med. 8:1145(2002)).

Accordingly, the present invention provides, in a further aspect,compounds of formulae (I), (IA), (IA′), (IB) and/or (IC) for use in thetreatment of hormone dependent cancers, such as breast and prostatecancers. By this use, it is aimed to reverse hormone resistance commonlyseen in these cancers with conventional anticancer agents.

In hematological cancers, such as chronic myelogenous leukemia (CML),chromosomal translocation is responsible for the constitutivelyactivated BCR-Abl tyrosine kinase. The afflicted patients are responsiveto imatinib, a small molecule tyrosine kinase inhibitor, as a result ofinhibition of Abl kinase activity. However, many patients with advancedstage disease respond to imatinib initially, but then relapse later dueto resistance-conferring mutations in the Abl kinase domain. In vitrostudies have demonstrated that BCR-Ab1 employs the Ras-Raf kinasepathway to elicit its effects. In addition, inhibiting more than onekinase in the same pathway provides additional protection againstresistance-conferring mutations.

Accordingly, in another aspect, the present invention provides thecompounds of formulae (I), (IA), (IA′), (IB) and/or (IC) for use incombination with at least one additional agent selected from the groupof kinase inhibitors, such as Gleevec®, in the treatment ofhematological cancers, such as chronic myelogenous leukemia (CML). Bythis use, it is aimed to reverse or prevent resistance to said at leastone additional agent.

Because activation of the PI3K/Akt pathway drives cell survival,inhibition of the pathway in combination with therapies that driveapoptosis in cancer cells, including radiotherapy and chemotherapy, willresult in improved responses (Ghobrial et al., C A Cancer J. Clin55:178-194 (2005)). As an example, combination of PI3 kinase inhibitorwith carboplatin demonstrated synergistic effects in both in vitroproliferation and apoptosis assays as well as in in vivo tumor efficacyin a xenograft model of ovarian cancer (Westfall and Skinner, Mol.Cancer Ther. 4:1764-1771 (2005)).

In addition to cancer and proliferative diseases, there is accumulatingevidence that inhibitors of Class 1A and 1B PI3 kinases would betherapeutically useful in others disease areas. The inhibition of p110β,the PI3K isoform product of the PIK3CB gene, has been shown to beinvolved in shear-induced platelet activation (Jackson et al., NatureMedicine 11:507-514 (2005)). Thus, a PI3K inhibitor that inhibits p110βwould be useful as a single agent or in combination in anti-thrombotictherapy. The isoform p110δ, the product of the PIK3CD gene, is importantin B cell function and differentiation (Clayton et al., J. Exp. Med.196:753-763 (2002)), T-cell dependent and independent antigen responses(Jou et al., Mol. Cell. Biol. 22:8580-8590 (2002)) and mast celldifferentiation (Ali et al., Nature 431:1007-1011 (2004)). Thus, it isexpected that p110δ-inhibitors would be useful in the treatment ofB-cell driven autoimmune diseases and asthma. Finally, the inhibition ofp110γ, the isoform product of the PI3KCG gene, results in reduced T, butnot B cell, response (Reif et al., J. Immunol. 173:2236-2240 (2004)) andits inhibition demonstrates efficacy in animal models of autoimmunediseases (Camps et al., Nature Medicine 11:936-943 (2005), Barber etal., Nature Medicine 11:933-935 (2005)).

The invention further provides pharmaceutical compositions comprising atleast one compound of formula (I), (IA), (IA′), (IB) and/or (IC),together with a pharmaceutically acceptable excepient suitable foradministration to a human or animal subject, either alone or togetherwith another therapeutic agent, for example another anticancer agent.

The invention further provides methods of treating human or animalsubjects suffering from a cellular proliferative disease, such ascancer. The invention thus provides methods of treating a human oranimal subject in need of such treatment, comprising administering tothe subject a therapeutically effective amount of a compound of formula(I), (IA), (IA′), (IB) and/or (IC) either alone or in combination withone or more other therapeutic agents, e.g. other anticancer agents. Inparticular, compositions will either be formulated together as acombination therapeutic or administered separately. Suitable anticanceragents for use with a compound of formula I include, but are not limitedto, one or more compounds selected from the group consisting of kinaseinhibitors, anti-estrogens, anti androgens, other inhibitors, cancerchemotherapeutic drugs, alkylating agents, chelating agents, biologicalresponse modifiers, cancer vaccines, agents for antisense therapy as setforth below:

A. Kinase Inhibitors:_Kinase inhibitors for use as anticancer agents inconjunction with a compound of the formula (I), (IA), (IA′), (IB) and/or(IC) include inhibitors of Epidermal Growth Factor Receptor (EGFR)kinases such as small molecule quinazolines, for example gefitinib (U.S.Pat. No. 5,457,105, U.S. Pat. No. 5,616,582, and U.S. Pat. No.5,770,599), ZD-6474 (WO 01/32651), erlotinib (Tarceva®, U.S. Pat. No.5,747,498 and WO 96/30347), and lapatinib (U.S. Pat. No. 6,727,256 andWO 02/02552); Vascular Endothelial Growth Factor Receptor (VEGFR) kinaseinhibitors, including SU-11248 (WO 01/60814), SU 5416 (U.S. Pat. No.5,883,113 and WO 99/61422), SU 6668 (U.S. Pat. No. 5,883,113 and WO99/61422), CHIR-258 (U.S. Pat. No. 6,605,617 and U.S. Pat. No.6,774,237), vatalanib or PTK-787 (U.S. Pat. No. 6,258,812), VEGF-Trap(WO 02/57423), B43-Genistein (WO-09606116), fenretinide (retinoic acidp-hydroxyphenylamine) (U.S. Pat. No. 4,323,581), IM-862 (WO 02/62826),bevacizumab or Avastin® (WO 94/10202), KRN-951,3-[5-(methylsulfonylpiperadine methyl)-indolyl]-quinolone, AG-13736 andAG-13925, pyrrolo[2,1-f][1,2,4]triazines, ZK-304709, Veglin®, VMDA-3601,EG-004, CEP-701 (U.S. Pat. No. 5,621,100), Cand5 (WO 04/09769); Erb2tyrosine kinase inhibitors such as pertuzumab (WO 01/00245),trastuzumab, and rituximab; Akt protein kinase inhibitors, such asRX-0201; Protein Kinase C (PKC) inhibitors, such as LY-317615 (WO95/17182), and perifosine (US 2003171303); Raf/Map/MEK/Ras kinaseinhibitors including sorafenib (BAY 43-9006), ARQ-350RP, LErafAON,BMS-354825 AMG-548, and others disclosed in WO 03/82272; FibroblastGrowth Factor Receptor (FGFR) kinase inhibitors; Cell Dependent Kinase(CDK) inhibitors, including CYC-202 or roscovitine (WO 97/20842 and WO99/02162); Platelet-Derived Growth Factor Receptor (PDGFR) kinaseinhibitors such as CHIR-258, 3G3 mAb, AG-13736, SU-11248 and SU6668; andBcr-Abl kinase inhibitors and fusion proteins such as STI-571 orGleevec® (imatinib).

B. Anti-Estrogens:_Estrogen-targeting agents for use in anticancertherapy in conjunction with a compound of formula (I), (IA), (IA′), (IB)and/or (IC) include Selective Estrogen Receptor Modulators (SERMs)including tamoxifen, toremifene, raloxifene; aromatase inhibitorsincluding Arimidex® or anastrozole; Estrogen Receptor Downregulators(ERDs) including Faslodex® or fulvestrant.

C. Anti-Androgens:_Androgen-targeting agents for use in anticancertherapy in conjunction with a compound of formula (I), (IA), (IA′), (IB)and/or (IC) include flutamide, bicalutamide, finasteride,aminoglutethamide, ketoconazole, and corticosteroids.

D. Other Inhibitors:_(—) Other inhibitors for use as anticancer agentsin conjunction with a compound of formula (I), (IA), (IA′), (IB) and/or(IC) include protein farnesyl transferase inhibitors includingtipifarnib or R-115777 (US 2003134846 and WO 97/21701), BMS-214662,AZD-3409, and FTI-277; topoisomerase inhibitors including merbarone anddiflomotecan (BN-80915); mitotic kinesin spindle protein (KSP)inhibitors including SB-743921 and MKI-833; proteasome modulators suchas bortezomib or Velcade® (U.S. Pat. No. 5,780,454), XL-784; andcyclooxygenase 2 (COX-2) inhibitors including non-steroidalantiinflammatory drugs I (NSAIDs).

E. Cancer Chemotherapeutic Drugs: Particular cancer chemotherapeuticagents for use as anticancer agents in conjunction with a compound offormula (I), (IA), (IA′), (IB) and/or (IC) include anastrozole(Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®),busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine(Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin(Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin(Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® orNeosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabineliposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®, US 2004073044), doxorubicin hydrochloride(Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate(Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®),tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea(Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan(Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan(Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®),mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix(Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustineimplant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®),6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecanhydrochloride for injection (Hycamptin®), vinblastine (Velban®),vincristine (Oncovin®), and vinorelbine (Navelbin®).

F. Alkylating Agents:_Alkylating agents for use in conjunction with acompound of formula (I), (IA), (IA′), (IB) and/or (IC) includeVNP-40101M or cloretizine, oxaliplatin (U.S. Pat. No. 4,169,846, WO03/24978 and WO 03/04505), glufosfamide, mafosfamide, etopophos (U.S.Pat. No. 5,041,424), prednimustine; treosulfan; busulfan; irofluven(acylfulvene); penclomedine; pyrazoloacridine (PD-115934);O6-benzylguanine; decitabine (5-aza-2-deoxycytidine); brostallicin;mitomycin C (MitoExtra); TLK-286 (Telcyta®); temozolomide; trabectedin(U.S. Pat. No. 5,478,932); AP-5280 (Platinate formulation of Cisplatin);porfiromycin; and clearazide (meclorethamine).

G. Chelating Agents:_Chelating agents for use in conjunction with acompound of formula (I), (IA), (IA′), (IB) and/or (IC) includetetrathiomolybdate (WO 01/60814); RP-697; Chimeric T84.66 (cT84.66);gadofosveset (Vasovist®); deferoxamine; and bleomycin optionally incombination with electorporation (EPT).

H. Biological Response Modifiers:_Biological response modifiers, such asimmune modulators, for use in conjunction with a compound of formula(I), (IA), (IA′), (IB) and/or (IC) include staurosprine and macrocyclicanalogs thereof, including UCN-01, CEP-701 and midostaurin (see WO02/30941, WO 97/07081, WO 89/07105, U.S. Pat. No. 5,621,100, WO93/07153, WO 01/04125, WO 02/30941, WO 93/08809, WO 94/06799, WO00/27422, WO 96/13506 and WO 88/07045); squalamine (WO 01/79255);DA-9601 (WO 98/04541 and U.S. Pat. No. 6,025,387); alemtuzumab;interferons (e.g. IFN-a, IFN-b etc.); interleukins, specifically IL-2 oraldesleukin as well as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, and active biological variants thereof having aminoacid sequences greater than 70% of the native human sequence;altretamine (Hexalen®); SU 101 or leflunomide (WO 04/06834 and U.S. Pat.No. 6,331,555); imidazoquinolines such as resiquimod and imiquimod (U.S.Pat. Nos. 4,689,338, 5,389,640, 5,268,376, 4,929,624, 5,266,575,5,352,784, 5,494,916, 5,482,936, 5,346,905, 5,395,937, 5,238,944, and5,525,612); and SMIPs, including benzazoles, anthraquinones,thiosemicarbazones, and tryptanthrins (WO 04/87153, WO 04/64759, and WO04/60308).

I. Cancer Vaccines:_Anticancer vaccines for use in conjunction with acompound of formula (I), (IA), (IA′), (IB) and/or (IC) include Avicine®(Tetrahedron Lett. 26:2269-70 (1974)); oregovomab (OvaRex®); Theratope®(STn-KLH); Melanoma Vaccines; GI-4000 series (GI-4014, GI-4015, andGI-4016), which are directed to five mutations in the Ras protein;GlioVax-1; MelaVax; Advexin® or INGN-201 (WO 95/12660); Sig/E7/LAMP-1,encoding HPV-16 E7; MAGE-3 Vaccine or M3TK (WO 94/05304); HER-2VAX;ACTIVE, which stimulates T-cells specific for tumors; GM-CSF cancervaccine; and Listeria monocytogenes-based vaccines.

J. Antisense Therapy:_Anticancer agents for use in conjunction with acompound of formula (I), (IA), (IA′), (IB) and/or (IC) also includeantisense compositions, such as AEG-35156 (GEM-640); AP-12009 andAP-11014 (TGF-beta2-specific antisense oligonucleotides); AVI-4126;AVI-4557; AVI-4472; oblimersen (Genasense®); JFS2; aprinocarsen (WO97/29780); GTI-2040 (R2 ribonucleotide reductase mRNA antisense oligo)(WO 98/05769); GTI-2501 (WO 98/05769); liposome-encapsulated c-Rafantisense oligodeoxynucleotides (LErafAON) (WO 98/43095); and Sirna-027(RNAi-based therapeutic targeting VEGFR-1 mRNA).

A compound of formula (I), (IA), (IA′), (IB) and/or (IC) may also becombined in a pharmaceutical composition with bronchiodilatory orantihistamine drugs substances. Such bronchiodilatory drugs includeanticholinergic or antimuscarinic agents, in particular glycopyrrolate,ipratropium bromide, oxitropium bromide, and tiotropium bromide, OrM3,aclidinium, CHF5407, GSK233705 and β-2-adrenoreceptor agonists such assalbutamol, terbutaline, salmeterol, carmoterol, milveterol and,especially, indacaterol and formoterol. Co-therapeutic antihistaminedrug substances include cetirizine hydrochloride, clemastine fumarate,promethazine, loratadine, desloratadine diphenhydramine and fexofenadinehydrochloride.

The invention provides in a further aspect a combination comprising acompound of formula (I), (IA), (IA′), (IB) and/or (IC) and one or morecompounds that are useful for the treatment of a thrombolytic disease,heart disease, stroke, etc. Such compounds include aspirin, astreptokinase, a tissue plasminogen activator, a urokinase, aanticoagulant, antiplatelet drugs (e.g, PLAVIX; clopidogrel bisulfate),a statin (e.g., LIPITOR or Atorvastatin calcium), ZOCOR (Simvastatin),CRESTOR (Rosuvastatin), etc.), a Beta blocker (e.g., Atenolol), NORVASC(amlodipine besylate), and an ACE inhibitor (e.g., lisinopril).

The invention provides in a further aspect a combination comprising acompound of formula (I), (IA), (IA′), (IB) and/or (IC) and one or morecompounds that are useful for the treatment of antihypertension. Suchcompounds include ACE inhibitors, lipid lowering agents such as statins,LIPITOR (Atorvastatin calcium), calcium channel blockers such as NORVASC(amlodipine besylate).

The invention provides in a further aspect a combination comprising acompound of formula (I), (IA), (IA′), (IB) and/or (IC) and one or morecompounds selected from the group consisting of fibrates, beta-blockers,NEPI inhibitors, Angiotensin-2 receptor antagonists and plateletaggregation inhibitors.

The invention provides in a further aspect a combination comprising acompound of formula (I), (IA), (IA′), (IB) and/or (IC) and a compoundsuitable for the treatment of inflammatory diseases, includingrheumatoid arthritis. Such compound may be selected from the groupconsisting of TNF-α inhibitors such as anti-TNF-α monoclonal antibodies(such as REMICADE, CDP-870) and D2E7 (HUMIRA) and TNF receptorimmunoglobulin fusion molecules (such as ENBREL), IL-1 inhibitors,receptor antagonists or soluble IL-1R

(e.g. KINERET or ICE inhibitors), nonsterodial anti-inflammatory agents(NSAIDS), piroxicam, diclofenac, naproxen, flurbiprofen, fenoprofen,ketoprofen ibuprofen, fenamates, mefenamic acid, indomethacin, sulindac,apazone, pyrazolones, phenylbutazone, aspirin, COX-2 inhibitors (such asCELEBREX (celecoxib), PREXIGE (lumiracoxib)), metalloprotease inhibitors(preferably MMP-13 selective inhibitors), p2×7 inhibitors, α2

inhibitors, NEUROTIN, pregabalin, low dose methotrexate, leflunomide,hydroxyxchloroquine, d-penicillamine, auranofin or parenteral or oralgold.

The invention provides in a further aspect a combination comprising acompound of formula (I), (IA), (IA′), (IB) and/or (IC) and a compoundsuitable for the treatment of osteoarthritis. Such compound may beselected from the group consisting of standard non-steroidalanti-inflammatory agents (hereinafter NSAID's) such as piroxicam,diclofenac, propionic acids such as naproxen, flurbiprofen, fenoprofen,ketoprofen and ibuprofen, fenamates such as mefenamic acid,indomethacin, sulindac, apazone, pyrazolones such as phenylbutazone,salicylates such as aspirin, COX-2 inhibitors such as celecoxib,valdecoxib, lumiracoxib and etoricoxib, analgesics and intraarticulartherapies such as corticosteroids and hyaluronic acids such as hyalganand synvisc.

The invention provides in a further aspect a combination comprising acompound of formula (I), (IA), (IA′), (IB) and/or (IC) and an antiviralagent and/or an antisepsis compound. Such antiviral agent may beselected from the group consisting of Viracept, AZT, acyclovir andfamciclovir. Such antisepsis compound may be selected from the groupconsisting of Valant.

The invention provides in a further aspect a combination comprising acompound of formula (I), (IA), (IA′), (IB) and/or (IC) and one or moreagents selected from the group consisting of CNS agents such asantidepressants (sertraline), anti-Parkinsonian drugs (such as deprenyl,L-dopa, Requip, Mirapex; MAOB inhibitors (such as selegine andrasagiline); comP inhibitors (such as Tasmar); A-2 inhibitors; dopaminereuptake inhibitors; NMDA antagonists; Nicotine agonists; Dopamineagonists; and inhibitors of neuronal nitric oxide synthase).

The invention provides in a further aspect a combination comprising acompound of formula (I), (IA), (IA′), (IB) and/or (IC) and one or moreanti-Alzheimer's drugs. Such anti-Alzheimer Drug may be selected fromthe group consisting of donepezil, tacrine, α2δinhibitors, NEUROTIN,pregabalin, COX-2 inhibitors, propentofylline or metryfonate.

The invention provides in a further aspect a combination comprising acompound of formula (I), (IA), (IA′), (IB) and/or (IC) andanosteoporosis agents and/or an immunosuppressant agent. Suchosteoporosis agents ma be selected from the group consisting of EVISTA(raloxifene hydrochloride), droloxifene, lasofoxifene or fosomax. Suchimmunosuppressant agents may be selected from the group consisting ofFK-506 and rapamycin.

In another aspect of the preferred embodiments, kits that include one ormore compound of formula (I), (IA), (IA′), (IB) and/or (IC) and acombination partner as disclosed herein are provided. Representativekits include a PI3K inhibitor compound (e.g., a compound of formula (I),(IA), (IA′), (IB) and/or (IC)) and a package insert or other labelingincluding directions for treating a cellular proliferative disease byadministering a PI3K inhibitory amount of the compound(s).

In general, the compounds of formula (I), (IA), (IA′), (IB) and/or (IC)will be administered in a therapeutically effective amount by any of theaccepted modes of administration for agents that serve similarutilities. The actual amount of the compound of formula (I), (IA),(IA′), (IB) and/or (IC), i.e., the active ingredient, will depend uponnumerous factors such as the severity of the disease to be treated, theage and relative health of the subject, the potency of the compoundused, the route and form of administration, and other factors. The drugcan be administered more than once a day, preferably once or twice aday. All of these factors are within the skill of the attendingclinician. Therapeutically effective amounts of compounds of formulas Imay range from about 0.05 to about 50 mg per kilogram body weight of therecipient per day; preferably about 0.1-25 mg/kg/day, more preferablyfrom about 0.5 to 10 mg/kg/day. Thus, for administration to a 70 kgperson, the dosage range would most preferably be about 35-70 mg perday.

In general, compounds of formula (I), (IA), (IA′), (IB) and/or (IC) willbe administered as pharmaceutical compositions by any one of thefollowing routes: oral, systemic (e.g., transdermal, intranasal or bysuppository), or parenteral (e.g., intramuscular, intravenous orsubcutaneous) administration. The preferred manner of administration isoral using a convenient daily dosage regimen that can be adjustedaccording to the degree of affliction. Compositions can take the form oftablets, pills, capsules, semisolids, powders, sustained releaseformulations, solutions, suspensions, elixirs, aerosols, or any otherappropriate compositions. Another preferred manner for administeringcompounds of the formula I is inhalation. This is an effective methodfor delivering a therapeutic agent directly to the respiratory tract.

The choice of formulation depends on various factors such as the mode ofdrug administration and bioavailability of the drug substance. Fordelivery via inhalation the compound can be formulated as liquidsolution, suspensions, aerosol propellants or dry powder and loaded intoa suitable dispenser for administration. There are several types ofpharmaceutical inhalation devices-nebulizer inhalers, metered doseinhalers (MDI) and dry powder inhalers (DPI). Nebulizer devices producea stream of high velocity air that causes the therapeutic agents (whichare formulated in a liquid form) to spray as a mist that is carried intothe patient's respiratory tract. MDI's typically are formulationpackaged with a compressed gas. Upon actuation, the device discharges ameasured amount of therapeutic agent by compressed gas, thus affording areliable method of administering a set amount of agent. DPI dispensestherapeutic agents in the form of a free flowing powder that can bedispersed in the patient's inspiratory air-stream during breathing bythe device. In order to achieve a free flowing powder, the therapeuticagent is formulated with an excipient such as lactose. A measured amountof the therapeutic agent is stored in a capsule form and is dispensedwith each actuation.

The inventions also relates to formulations wherein the particle size ofa compound of formula I between 10-1000 nm, preferably 10-400 nm. Suchpharmaceutical formulations have been developed especially for drugsthat show poor bioavailability based upon the principle thatbioavailability can be increased by increasing the surface area i.e.,decreasing particle size. For example, U.S. Pat. No. 4,107,288 describesa pharmaceutical formulation having particles in the size range from 10to 1,000 nm in which the active material is supported on a crosslinkedmatrix of macromolecules. U.S. Pat. No. 5,145,684 describes theproduction of a pharmaceutical formulation in which the drug substanceis pulverized to nanoparticles (average particle size of 400 nm) in thepresence of a surface modifier and then dispersed in a liquid medium togive a pharmaceutical formulation that exhibits remarkably highbioavailability. Both documents are included by reference.

In a further aspect, the invention provides pharmaceutical compositionscomprising a (therapeutically effective amount) of a compound of formula(I), (IA), (IA′), (IB) and/or (IC), and at least one pharmaceuticallyacceptable excipient. Acceptable excipients are non-toxic, aidadministration, and do not adversely affect the therapeutic benefit ofthe compound of formula (I), (IA), (IA′), (IB) and/or (IC). Suchexcipient may be any solid, liquid, semi-solid or, in the case of anaerosol composition, gaseous excipient that is generally available toone of skill in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like.

Liquid and semisolid excipients may be selected from glycerol, propyleneglycol, water, ethanol and various oils, including those of petroleum,animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil,mineral oil, sesame oil, etc. Preferred liquid carriers, particularlyfor injectable solutions, include water, saline, aqueous dextrose, andglycols.

Compressed gases may be used to disperse a compound of the formula I inaerosol form. Inert gases suitable for this purpose are nitrogen, carbondioxide, etc. Other suitable pharmaceutical excipients and theirformulations are described in Remington's Pharmaceutical Sciences,edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).

The amount of the compound in a formulation can vary within the fullrange employed by those skilled in the art. Typically, the formulationwill contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt% of a compound of formula I based on the total formulation, with thebalance being one or more suitable pharmaceutical excipients.Preferably, the compound is present at a level of about 1-80 wt %.

The invention further relates to pharmaceutical compositions comprising(i.e. containing or consisting of) at least one compound of formula (I),(IA), (IA′), (IB) and/or (IC) and at least one pharmaceuticallyacceptable excipient.

Pharmaceutical compositions comprising a compound of formula (I), (IA),(IA′), (IB) and/or (IC) in free form or in pharmaceutically acceptablesalt form in association with at least one pharmaceutical acceptableexcipient (such as a carrier and/or diluent) may be manufactured inconventional manner by mixing the components.

Combined pharmaceutical compositions comprising a compound of formula(I), (IA), (IA′), (IB) and/or (IC) in free form or in pharmaceuticallyacceptable salt form and further comprising a combination partner(either in one dosage unit form or as a kit of parts) in associationwith at least one pharmaceutical acceptable carrier and/or diluent maybe manufactured in conventional manner by mixing with a pharmaceuticallyacceptable carrier and/or diluent with said active ingredients.

Consequently, the invention provides in further aspects

-   -   a combined pharmaceutical composition, e.g. for use in any of        the methods described herein, comprising a compound of formula        (I), (IA), (IA′), (IB) and/or (IC) in free form or        pharmaceutically acceptable salt form in association with a        pharmaceutically acceptable diluent and/or carrier.    -   a combined pharmaceutical composition comprising a compound of        formula (I), (IA), (IA′), (IB) and/or (IC) in free form or in        pharmaceutically acceptable salt form as active ingredient; one        or more pharmaceutically acceptable carrier material(s) and/or        diluents and optionally one or more further drug substances.        Such combined pharmaceutical composition may be in the form of        one dosage unit form or as a kit of parts.    -   a combined pharmaceutical composition comprising a        therapeutically effective amount of a compound of formula (I),        (IA), (IA′), (IB) and/or (IC) in free form or in        pharmaceutically acceptable salt form and a second drug        substance, for simultaneous or sequential administration.    -   a method as defined above comprising co-administration, e.g.        concomitantly or in sequence, of a therapeutically effective        non-toxic amount of a compound of formula (I), (IA), (IA′), (IB)        and/or (IC) or a pharmaceutically acceptable salt thereof, and        at least a second drug substance, e.g. as indicated above.    -   a pharmaceutical combination, e.g. a kit, comprising a) a first        agent which is a compound of formula (I), (IA), (IA′), (IB)        and/or (IC) as disclosed herein, in free form or in        pharmaceutically acceptable salt form, and b) at least one        co-agent, e.g. as indicated above; whereby such kit may comprise        instructions for its administration.

The following examples of compounds formula (I), (IA), (IA′), (IB)and/or (IC) illustrate the invention without limiting the scope thereof.Methods for preparing such compounds are described.

Temperatures are measured in degrees Celsius. Unless otherwiseindicated, the reactions take place at rt and the MS are obtained withESI. The following HPLC/MS methods are used in the preparation andanalysis of the Intermediates and Examples:

Methods A1 to A3 (LCMS: analytical HPLC/MS):

System: Agilent 1100 Series with Waters Micromass ZQ 2000 ESI+ and/orESI−

Column: XBridge C18, 3×30 mm, 2.5 micron

Temperature: 50° C.

Eluent A: H₂O, containing 5% CH₃CN and 0.8% HCOOH

Eluent B: CH₃CN, containing 0.6% HCOOH

Flow Rate: 1.2-2.4 mL/min

Method A1: method Polar4a_p_(—)100-900 and method Polar4a_pn_(—)100-900:

Gradient: 0-2.9 min: 1% to 95% of B

Method A2: method Fast4_p_(—)100-900 and method Fast4a_pn_(—)100-900:

Gradient: 0-2.4 min: 10% to 95% of B

Method A3: method Slow4a_pn_(—)100-900:

Gradient: 0-4.4 min: 5% to 95% of B

Method B (preparative HPLC) Instrument: Waters preparative HPLC system,column: Sunfire™ Prep C18 OBD™ 5 micron 30×100 mm, temperature: 25° C.,eluent: gradient from 5-100% CH₃CN in 0.05% aqueous TFA over 20 minutes,flow rate: 30 ml/minute, detection: UV 254 nm.

Method C (preparative HPLC) Instrument: Waters preparative HPLC system,column: Sunfire™ Prep C18 OBD™ 5 micron 30×100 mm, temperature: 25° C.,eluent: gradient from 5-50% CH₃CN in 0.05% aqueous TFA over 20 minutes,flow rate: 30 ml/minute, detection: UV 254 nm.

Method D (Analytical HPLC): Linear gradient 2-100% CH₃CN (0.1% TFA) andH₂O (0.1% TFA) in 5 min+1.5 min 100% CH₃CN (0.1% TFA); detection at 215nm, flow rate 1 mL/min at 30° C. Column: Nucleosil 100-3 C18 (70×4 mm)

Method E (preparative HPLC/MS) Instrument: Gilson preparative HPLCsystem, column: Sunfire™ Prep C18 OBD™ 5 micron 30×100 mm, temperature:25° C., eluent: gradient from 5-100% CH₃CN in 0.05% aqueous TFA over 20minutes, flow rate: 30 ml/minute, detection: UV 254 nm.

Method F (Analytical HPLC) Instrument: Shimadzu SIL-10A, Method: Lineargradient 2-100% CH₃CN (0.1% TFA) and H₂O (0.1% TFA) in 4 min+2 min 100%CH₃CN (0.1% TFA); back to −100% CH₃CN (0.1% TFA) in 3 min.; detection at215 nm, flow rate 2 mL/min at RT. Column: Nucleosil OD-5-100 C18(150×4.6 mm)

Method G (Analytical HPLC) Instrument:

System: Agilent 1100 Series

Column: HP Hypersil BDS C18, 4×125 mm, 5 micron

Temperature: 25° C.

Eluent A: H₂O, containing 0.1% v/v TFA

Eluent B: CH₃CN, containing 0.1% v/v TFA

Gradient: 10%→100% B in 5 min, 2.5 min with 100% B, then→10% B in 1 min

Flow Rate: 1.5 mL/min

Detection: UV 215 nm

Method H (Analytical HPLC) Instrument:

System: Agilent 1100 Series

Column: Macherey-Nagel Nucleosil 100-3 C18HD, 4×125 mm, 3 micron

Temperature: 30° C.

Eluent A: H₂O, containing 0.1% v/v TFA

Eluent B: CH₃CN, containing 0.1% v/v TFA

Gradient: 2%→100% B in 7 min, 2 min with 100% B, then→2% B in 1 min

Flow Rate: 1.0 mL/min

Detection: UV 215 nm

Method I (LCMS: analytical HPLC/MS):

System: Waters Acquity UPLC with Waters Micromass ZQ 2000 ESI+/−

Column: Acquity HSS T3 C18, 2.1×50 mm, 1.8 micron

Temperature: 50° C.

Eluent A: H₂O, containing 0.05% v/v HCOOH and 3.75 mM ammonium acetate

Eluent B: CH₃CN, containing 0.04% HCOOH

Gradient: 2%→98% B in 4.3 min, 0.7 min with 98% B, then→2% B in 0.1 minand 0.9 min with 2% B

Flow Rate: 1.0 mL/min

Method J (LCMS: analytical HPLC/MS):

System: Agilent 1100 Series; MS: G1946D

Column: Symmetry C8, 2.1×50 mm, 3.5 micron

Eluent A: H₂O, containing 0.1% v/v HCOOH

Eluent B: CH₃CN, containing 0.1% v/v HCOOH

Gradient: 0-3.3 min: 5% to 95% of B

Flow Rate: 1.0 mL/min

Method K (LCMS: analytical HPLC/MS):

System: Waters Acquity UPLC

Column: Acquity HSS T3 C18, 2.1×50 mm, 1.8 micron

Eluent A: H₂O, containing 0.05% v/v HCOOH and 0.05% ammonium acetate

Eluent B: acetonitrile, containing 0.04% HCOOH

Gradient: 2%→98% B in 1.7 min, 0.45 min with 98% B, then→2% B in 0.04min

Flow Rate: 1.2 ml/min

Method L (LCMS: analytical HPLC/MS):

System: Waters Aquity UPLC; MS: Waters AQ Detector

Column: Aquity HSS, 1.8 μm 2.1×50 mm, 3/pk

Eluent A: H₂O, containing 0.1% v/v HCOOH

Eluent B: CH₃CN, containing 0.1% v/v HCOOH

Gradient: 0-1.5 min: 10% to 95% of B, then 1 min: 95% B

Flow Rate: 1.2 mL/min

ESI-MS:

Instrument: Micromass Platform II

Eluent: 15% v/v MeOH in H₂O containing 0.2% v/v of a 25% ammoniumhydroxide solution

Flow Rate: 0.05 mL/min

In the following examples, the abbreviations given below are used:

-   -   atm. atmosphere    -   CDI 1,1′-carbonyldiimidazole    -   CH₃CN acetonitrile    -   DAST diethylaminosulfur trifluoride    -   DCE 1,2-dichloroethane    -   DCM dichloromethane    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulfoxide    -   Et₂O diethyl ether    -   EtOAc ethyl acetate    -   EtOH ethanol    -   eq equivalent(s)    -   h hour(s)    -   H₂O water    -   HPLC High Performance Liquid Chromatography    -   HV high vacuum    -   LCMS liquid chromatography coupled with mass spectrometry    -   LiHMDS lithium bis(trimethylsilyl)amide    -   MeOH methanol    -   mL milliliter(s)    -   min minute(s)    -   MS-ESI electrospray ionisation mass spectrometry    -   MW microwave    -   NaHCO₃ sodium bicarbonate    -   Na₂SO₄ sodium sulfate    -   NH₄Cl ammonium chloride    -   RM reaction mixture    -   R_(f) ratio of fronts in TLC    -   rt room temperature    -   TEA triethylamine    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran    -   TLC thin layer chromatography    -   t_(R) retention time    -   UV Ultraviolet

Intermediate A: Imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide

8-tert-Butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-ylamine (Stage A.1,0.319 g, 1.225 mmol) and CDI (278 mg, 1.715 mmol) were added to DCM (5ml) and DMF (0.25 ml) under an argon atm. After 18 h the residue wascooled to 4° C. and the precipitate was collected by filtration. Thesolid was dried at 50° C. in high vacuo to give the title compound as apale yellow solid. LCMS: t_(R) 0.95 min and M+H 319.0 (method A2) for(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-carbamic acidmethyl ester; the product of the reaction of the title compound withMeOH during the preparation of the sample as MeOH solution.

Stage A.1: 8-tert-Butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-ylamine

Intermediate A.1 was obtained by two different routes. Both routes startfrom 2-amino-5,6-dihydro-4H-benzothiazol-7-one and described below:

Route 1:

To a mixture ofN′-{6-[1-dimethylamino-meth-(E)-ylidene]-7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl}-N,N-dimethyl-formamidine(Stage A.2, 2.2 g, 7.90 mmol) in 2-methoxyethanol (20 mL) was added atrt sodium hydroxide (1.185 g, 29.6 mmol) and 2,2-dimethyl-propionamidinehydrochloride (1.620 g, 11.85 mmol). The RM was stirred at 125° C. for 3h. After cooling to rt, the RM was diluted with MeOH, adsorbed ontosilica gel and purified by flash chromatography (CombiFlash® Companionsystem®, with RediSep® silica gel column, eluent: DCM/MeOH/Ammonia95:5:0.5). LC: t_(R) 3.64 min (method D). MS: M+H=261. 1H-NMR inDMSO-d₆: 8.24 (s, 1H); 7.70 (s, 2H); 2.89-2.84 (m, 2H); 2.76-2.71 (m,2H); 1.28 (s, 9H).

Stage A.2:N′-{6-[1-Dimethylamino-meth-(E)-ylidene]-7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl}-N,N-dimethyl-formamidine

A suspension of 2-amino-5,6-dihydro-4H-benzothiazol-7-one (3.5 g, 20.81mmol) in dimethoxymethyldimethylamine (12 mL, 90 mmol) was heated at100° C. with stirring for 65 h. The RM was then evaporated to dryness invacuo and the residue was suspended in EtOAc. After 1 h at 4° C., thesolid was filtered off, washed with EtOAc and then dried under highvacuum at 60° C. to give the pure title product as brown crystals. LC:t_(R) 3.25 min (method D). MS: M+H=279. ¹H-NMR in DMSO-d₆: 8.40 (s, 1H);7.23 (s, 1H); 3.15 (s, 3H); 3.05 (s, 6H); 2.97 (s, 3H); 2.91 (t, 2H);2.67 (t, 2H).

Route 2:

Potassium carbonate (0.434 g, 3.14 mmol) was added to a mixture ofN-(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-acetamide(Stage A.3, 0.38 g, 1.257 mmol) in MeOH. The RM was stirred at 50° C.for 52 h, then cooled to rt and evaporated in vacuo to give a red mass.Water (20 mL) was added and the mixture was stirred at it for a further3 h. The red suspension was then cooled down to 4° C. and filtered togive after drying under high vacuum the title compound as a beige solid.LCMS: t_(R) 0.99 min and M+H=261 (method A3). ¹H-NMR in DMSO-d₆, 400MHz: 8.24 (s, 1H); 7.70 (s, 2H); 2.89-2.84 (m, 2H); 2.76-2.71 (m, 2H);1.28 (s, 9H).

Stage A.3:N-(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-acetamide

Pyridine (13 mL) was added toN-(6-formyl-7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide (StageA.4, 3.05 g, 12.8 mmol) and tert.butylamidine hydrochloride (1.788 g,12.8 mmol) and the mixture was heated in a sealed vessel at 160° C. for6.5 h. After cooling the reaction mixture was filtered to give a solid.The filtration mother liquor was evaporated to give further solidmaterial. The combined solids were triturated repeatedly with hot CH₃CNand the CH₃CN mother liquors evaporated to give a solid which was shownto be predominantly the title product. The crude product was dissolvedin ca. 10 ml of a 10% DMSO in MeOH to give a slightly hazy orangesolution which was filtered and added dropwise to water (100 ml) at itwith stirring. The precipitate solid was collected by filtration to givethe title compound as an orange solid. LCMS: t_(R) 1.37 min andM+H=303.0 (method A3). ¹H-NMR in DMSO-d₆, 400 MHz: 12.40 (s, 1H); 8.44(s, 1H); 2.88-3.00 (m, 4H); 2.17 (s, 3H); 1.32 (s, 9H).

Stage A.4:N-(6-Formyl-7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide

LiHMDS solution (1 M, 27.7 mL) was added over 10 min to a suspension ofN-(7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide (Stage A.5, 2.0g, 9.23 mmol) in dry THF (20 mL) cooled at −78° C. under an argon atm.The RM was then stirred at −78° C. for 2.5 h and methyl formate (2.308mL, 36.9 mmol) added dropwise over 30 min. The RM was then warmed slowlyto rt and was then stirred 18 h at rt. The RM was drown out into aqueous1 M HCl (70 mL) and extracted 3× with DCM, dried over Na₂SO₄ andevaporated to give a the title compound as a solid. HPLC: t_(R) 3.65 min(method D). MS: M−H=237. ¹H-NMR in DMSO-d₆ (400 MHz): 12.50 (s, br, 1H);7.55 (s, 1H); 2.90-2.60 (m, 4H); 2.15 (s, 3H).

Stage A.5: N-(7-Oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide

To acetic anhydride (80 mL) was added at rt2-amino-5,6-dihydro-4H-benzothiazol-7-one (10 g, 59.4 mmol) and theresulting suspension was heated to reflux. After 1.75 h stirring atreflux, the RM was allowed to cool with stirring and stirred for 18 h atit before further cooling with an ice/NaCl bath, and a solid wascollected by filtration. The solid was then triturated twice withrefluxing acetone (10 mL then 15 mL) before filtering and drying undervacuum at 40° C. to give the title product as a beige solid. HPLC: t_(R)3.47 min (method D). MS: M−H=211.1. ¹H-NMR in DMSO-d₆ (600 MHz): 12.55(s, br, 1H); 2.84 (t, 2H); 2.48 (t, 2H); 2.17 (s, 3H); 2.065 (qt, 2H).

Intermediate B: (2S,4R)-4-Dimethylamino-pyrrolidine-2-carboxylic acidamide

A solution of (2S,4R)-4-dimethylamino-pyrrolidine-2-carboxylic acidmethyl ester (Stage B.1, 225 mg) and 7 M ammonia in MeOH (7 mL) wasstood for 18 h at it in a sealed vessel. Evaporation and triturationwith Et₂O gave the title compound as a white solid.

Stage B.1 (2S,4R)-4-Dimethylamino-pyrrolidine-2-carboxylic acid methylester

A mixture of (2S,4R)-4-dimethylamino-pyrrolidine-1,2-dicarboxylic acid1-benzyl ester 2-methyl ester (Stage B.2, 420 mg), 10% palladium oncarbon (80 mg) and MeOH (10 mL) was stirred for 16 h under an atm. ofhydrogen. Filtration and evaporation gave the title compound which wasused without purification in the following steps.

Stage B.2 (2S,4R)-4-Dimethylamino-pyrrolidine-1,2-dicarboxylic acid1-benzyl ester 2-methyl ester

Sodium cyanoborohydride (200 mg) was added to a mixture of(2S,4R)-4-amino-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester2-methyl ester (400 mg), formalin (0.68 ml), acetic acid (0.72 ml),triethylamine (0.2 ml) and MeOH (2 ml) and the mixture was stirred for 2h at rt. The RM was then partitioned between DCM and aqueous NaHCO₃solution, the DCM layers evaporated and purified by normal phasechromatography, eluent; gradient from EtOAc to 20% EtOH in EtOAc, togive the predominant UV-active component. The chromatographied materialwas taken up with 1 M HCl, washed 2× with Et₂O, the aqueous layerbasified with NaHCO₃, 3× extracted with Et₂O, dried over Na₂SO₄ andevaporated to give the title compound as a pale yellow oil.

Intermediate C: Imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide

To a mixture of2-amino-8-N,N-diethylamino-4,5-dihydrothiazolo[4,5-h]quinazoline (StageC.1, 1 g, 3.63 mmol) in DCM (35 mL) was added CDI (1.178 g, 7.26 mmol).The RM was stirred at 40° C. for 90 h. After cooling to rt, the solidwas collected by filtration to give the title compound. HPLC: t_(R) 4.11min (method D). MS: M+H=334 for(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-carbamicacid methyl ester as the product of the reaction of the title compoundwith MeOH.

Stage C.1: 2-Amino-8-diethylamino-4,5-dihydrothiazolo[4,5-h]quinazoline

ToN′-{6-[1-dimethylamino-meth-(E)-ylidene]-7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl}-N,N-dimethyl-formamidine(Stage A.2, 1 g, 3.59 mmol) in 2-methoxyethanol (10 mL) was added NaOH(0.539 g, 13.47 mmol) and N,N-diethylguanidine (0.454 g, 3.94 mmol)under argon at rt. The RM was stirred for 3.5 h at 125° C. and thencooled to rt. After evaporation in vacuo the residue was dissolved in0.1 M HCl (50 mL) and washed with EtOAc. The aqueous layer was thenbasified with 6 N NaOH and extracted 3× with EtOAc. the organic layerswere dried over Na₂SO₄, evaporated and dried under high vacuum at 60° C.to give the title compound as orange crystals. LC: t_(R) 3.60 min(method D). MS: M+H=276. 1H-NMR in DMSO-d6: 7.91 (s, 1H); 7.59 (s, 2H);3.50 (q, 4H); 2.69 (dd, 4H); 1.07 (t, 6H).

Intermediate D: (2S,3S)-3-Methyl-pyrrolidine-2-carboxylic acid amide

A 4 M solution of HCl in 1,4-dioxan (1.5 mL) was added to a suspensionof (2S,3S)-3-methylpyrrolidine-2-carboxylic acid (0.5 g) in EtOH (5 mL)at rt and the mixture heated at reflux for 20 h. The RM was evaporatedand a 7 M solution of ammonia in MeOH (5.6 mL) added. The RM was stoodat it for 6 days then evaporated, the residue triturated with MeOH (0.5mL) and filtered and washed with cold MeOH (2 mL) to give the titlecompound as a white solid. ¹H-NMR (d₆-DMSO, 400 MHz): 8.06 (s, 1H), 7.67(s, 1H), 3.60 (d, 1H), 3.25-3.14 (m, 2H), 2.24-2.15 (m, 1H), 2.09-1.98(m, 1H), 1.57-1.45 (m, 1H), 1.13 (d, 3H).

Intermediate E: (R)-2-Benzyl-pyrrolidine-2-carboxylic acid amide

A mixture of(3R,7aR)-7a-benzyl-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1one(1.40 g, prepared as described by Wang and Germanas Synlett 1999,33-36.) and 7 M ammonia in MeOH (15 ml) was heated at 50° C. for 3 daysin a sealed vessel. The cooled RM was then evaporated and trituratedwith chloroform to give the title compound as a white solid.

Intermediate F (S)-2-Methyl-pyrrolidine-2-carboxylic acid amide

A solution of (S)-2-methyl-pyrrolidine-2-carboxylic acid butyl ester(Stage F.1, 2.3 g) in a 7 M solution of ammonia in MeOH (22.2 ml) washeated in a bomb at 70° C. for 10 days. Evaporation of the RM andtrituration with hexanes (20 mL) gave the title compound as an off-whitesolid. ¹H-NMR (d₆-DMSO, 400 MHz): 7.40 (s, 1H), 6.89 (s, 1H), 2.95-2.84(m, 1H), 2.72-2.60 (m, 1H), 2.06-1.95 (m, 1H), 1.66-1.44 (m, 2H),1.42-1.30 (m, 1H), 1.22 (s, 3H).

Stage F.1: (S)-2-Methyl-pyrrolidine-2-carboxylic acid butyl ester

Concentrated HCl (2 ml) was added to a suspension of(S)-2-methyl-pyrrolidine-2-carboxylic acid (2 g) in butan-1-ol (50 ml)which was heated at 60° C. for 18 h then at reflux for 4 days. The RMwas evaporated, partitioned between saturated aqueous NaHCO₃ and DCM,extracted 3× with DCM, dried over Na₂SO₄ and evaporated. The isolatedoil was then kugelrohr distilled at 10 mbar to give the title compoundas a clear colorless oil from the fraction distilling at an oventemperature of 100-120° C.

Intermediate G: (R)-2-Methoxymethyl-pyrrolidine-2-carboxylic acid amide

A mixture of(3R,7aR)-7a-methoxymethyl-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one(Stage G.1, 0.6 g) and 7 M ammonia in MeOH (6 mL) was stood at rt for 2days in a sealed vessel. The RM was then evaporated to give the titlecompound as a pale yellow oil which was used without furtherpurification.

Stage G.1:(3R,7aR)-7a-Methoxymethyl-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one

A 1M of solution of lithium diisopropylamide in a 3:5 mixture ofhexanes/THF (8.25 ml) was added dropwise to(3R,7aS)-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one (1.51g, prepared as described by Wang and Germanas Synlett 1999, 33-36.) inTHF (5 ml) at −78° C. After stirring 30 minutes at −78° C.methoxymethylchloride (1.14 ml) was added. The RM was then allowed towarm to −30° C. over 3 h and water was added. The aqueous layer wasextracted with DCM, the combined organic layers evaporated and theresidue was then purified by normal phase chromatography, eluting withDCM, to give the title compound as a pale yellow oil.

Intermediate H: (R)-2-Dimethylaminomethyl-pyrrolidine-2-carboxylic acidamide

A mixture of(3R,7aR)-7a-dimethylaminomethyl-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one(Stage H.1, 0.26 g) and 7 M ammonia in MeOH (4 mL) was heated at 50° C.for 3 days in a sealed vessel. The cooled RM was then evaporated to givethe title compound as a brown oil which was used without furtherpurification. MS: M+H=172.1.

Stage H.1:(3R,7aR)-7a-Dimethylaminomethyl-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one

A 1 M of solution of lithium diisopropylamide in a 3:5 mixture ofhexanes/THF (8.25 ml) was added dropwise to(3R,7aS)-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one (1.51g, prepared as described by Wang and Germanas Synlett 1999, 33-36.) inTHF (5 ml) at −78° C. After stirring 30 minutes at −78° C. Eschenmoser'ssalt (2.78 g) was added. The RM was then allowed to warm to −40° C. withvigorous stirring over 1 h and maintained for 2 h at −40° C. Water wasthen added and the aqueous layer extracted with DCM, the combinedorganic layers dried over Na₂SO₄ and evaporated. The residue was thenpurified by normal phase chromatography eluting with a gradient from DCMto 20% EtOAc in DCM to give the title compound as a pale yellow oil(M+H=301/303/305 3:3:1).

Intermediate I: d₆-(R)-2-Dimethylaminomethyl-pyrrolidine-2-carboxylicacid amide

d₆-(3R,7aR)-7a-Dimethylaminomethyl-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one(Stage I.1, 440 mg, 1.430 mmol) was dissolved in ammonia in MeOH (10.2mL, 71.4 mmol) in a sealed vessel and heated at 75° C. for 5 days. Thereaction mixture was evaporated and triturated 2× with CHCl₃ to give thetitle product as a pale brown solid.

Stage I.1:d₆-(3R,7aR)-7a-Dimethylaminomethyl-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one

(3R,7aR)-1-Oxo-3-trichloromethyl-dihydro-pyrrolo[1,2-c]oxazole-7a-carbaldehyde(obtained as described in J. Org. Chem. 2006, 71(1), 97-102, 1 g, 3.67mmol), dimethylamine-d₇ in THF (1.0 mL, 14.09 mmol), acetic acid (0.525mL, 9.17 mmol) and DCE (4 mL) were combined under argon and sodiumtriacetoxyborohydride (1.089 g, 5.14 mmol) was added portionwise. Afterstirring at it for 3 h the RM was taken up in DCM, and partitioned with1 M NaOH extracting once more with DCM. The organic layers were combinedthen washed twice with water before being dried over Na₂SO₄. Thesolution was evaporated to half of its volume and 1 M HCl in H₂O wasadded, the layers separated and the aqueous layer washed twice with DCM.The pH of the aqueous layers were adjusted to ˜8 with saturated Na₂CO₃solution and extracted three times with DCM. The organic layers weredried over Na₂SO₄ and evaporated, to give the desired title product as apale yellow oil.

Intermediate J: (R)-2-Hydroxymethyl-pyrrolidine-2-carboxylic acid amide

(3R,7aR)-7a-Hydroxymethyl-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one(Stage J.1, 308 mg, 1.122 mmol) was added to 7 M ammonia in MeOH (8.014mL, 56.1 mmol) under argon and heated at 75° C. in a sealed vessel for72 h and then evaporated to give the title compound as a viscous palebrown oil which was used without further purification.

Stage J.1:(3R,7aR)-7a-Hydroxymethyl-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one

Sodium triacetoxyborohydride (544 mg, 2.57 mmol) was added to(3R,7aR)-1-oxo-3-trichloromethyl-dihydro-pyrrolo[1,2-c]oxazole-7a-carbaldehyde(obtained as described in J. Org. Chem. 2006, 71(1), 97-102, 500 mg,1.835 mmol) in DCE (4 mL). The RM was stirred 18 h at rt then taken upin DCM, the organic layer washed with water, dried over Na₂SO₄ andevaporated. The crude product was purified using a 20 g RediSep® silicagel column (eluent DCM to 10% MeOH in DCM) to give the title compound asa clear pale yellow oil.

Intermediate K:(R)-2-{[(3-Fluoro-benzyl)-methyl-amino]-methyl}-pyrrolidine-2-carboxylicacid amide

(3R,7aR)-7a-{[(3-Fluoro-benzyl)-methyl-amino]-methyl}-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one(Stage K.1, 385 mg, 0.973 mmol) was added to 7 M ammonia in MeOH (6.950mL, 48.7 mmol) under argon and heated in a sealed vessel at 50° C. for 6days and then at 75° C. for 5 days. The RM was then evaporated and theresidue purified by flash chromatography using a 12 g RediSep® silicagel column (eluent 5% MeOH in DCM) to give the title compound as ayellow oil.

Stage K.1:(3R,7aR)-7a-{[(3-Fluoro-benzyl)-methyl-amino]-methyl}-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one

Sodium triacetoxyborohydride (889 mg, 4.19 mmol) was added to a mixtureof(3R,7aR)-7a-[(3-fluoro-benzylamino)-methyl]-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one(Stage K.2, 400 mg, 1.048 mmol), formaldehyde (37% in H₂O, 0.102 mL,1.36 mmol) and acetic acid (0.150 mL, 2.62 mmol) in DCE (4 mL) under anargon atm. After 2 h stirring at rt the RM was partitioned between DCMand water. The combined organic layers were dried over Na₂SO₄ andevaporated to give the title compound as a pale brown oil which was usedwithout further purification.

Stage K.2:(3R,7aR)-7a-[(3-Fluoro-benzylamino)-methyl]-3-trichloromethyl-tetrahydro-pyrrolo[1,2-c]oxazol-1-one

Sodium triacetoxyborohydride (544 mg, 2.57 mmol) was added to(3R,7aR)-1-oxo-3-trichloromethyl-dihydro-pyrrolo[1,2-c]oxazole-7a-carbaldehyde(obtained as described in J. Org. Chem. 2006, 71(1), 97-102, 500 mg,1.835 mmol), 3-fluorobenzylamine (0.251 mL, 2.20 mmol) and acetic acid(0.263 mL, 4.59 mmol) in DCE (4 mL) under an argon atm. The RM wasstirred at rt for 3 h, then partitioned between water and DCM, thecombined organic dried over Na₂SO₄ and evaporated to give the titlecompound as a pale yellow oil which was used without furtherpurification.

Intermediate L: (S)-Azetidine-2-carboxylic acid amide

A mixture of (S)-2-carbamoyl-azetidine-1-carboxylic acid benzyl ester(Stage L.1, 1.8 g) and 10% palladium on carbon (0.2 g) in MeOH (25 ml)was stirred under a hydrogen atm. at rt for 5 h. Filtration andevaporation gave the title compound which was used without furtherpurification.

Stage L.1: (S)-2-Carbamoyl-azetidine-1-carboxylic acid benzyl ester

A mixture of (S)-azetidine-1,2-dicarboxylic acid 1-benzyl ester 2-methylester (2.5 g) and 7 M ammonia in MeOH (10 ml) was stood at it for 18 hin a sealed vessel. The RM was then evaporated to give the titlecompound as a white solid which was used without further purification.MS: M+H 235.1 and M−H 233.1.

Intermediate M: (2S,4R)-4-fluoro-pyrrolidine-2-carboxylic acid amide

A 1.25 M solution of HCl in EtOH (2.3 ml) was added to a suspension of(2S,4R)-4-fluoro-pyrrolidine-2-carboxylic acid (0.25 g) in EtOH (2 ml)at rt and the mixture heated for 62 h at 55° C. The RM was evaporatedand a 7 M solution of ammonia in MeOH (5.6 ml) added. The RM was stoodat rt for 36 h then evaporated, the residue triturated with MeOH (0.5ml) and filtered to give the title compound as a white solid. ¹H-NMR(d₆-DMSO, 400 MHz): 7.68 (s, 1H), 7.37 (s, 1H), 5.30 (d, 1H), 3.96 (t,1H), 3.40-3.12 (m, 2H), 2.48-2.31 (m, 1H), 2.02-1.81 (m, 1H).

Intermediate N: (2S,4S)-4-Fluoro-pyrrolidine-2-carboxylic acid amide

A mixture of (2S,4S)-2-carbamoyl-4-fluoro-pyrrolidine-1-carboxylic acidtert-butyl ester (1.0 g), conc. HCl (0.6 ml) and 1-butanol (10 ml) washeated for 48 h at 50° C. The RM was evaporated and partitioned DCM andaqueous NaHCO₃, the DCM layers were dried over Na₂SO₄ and evaporated. Asolution of 7 M ammonia in methanol (10 ml) was added to the residue andthe mixture was stood for 60 h at it in a sealed vessel. Evaporation andtrituration with EtOH gave the title compound as a white solid.

Intermediate O: (1S,5R)-2-Aza-bicyclo[3.1.0]hexane-1-carboxylic acidamide

A mixture of (1S,5R)-2-aza-bicyclo[3.1.0]hexane-1-carboxylic acid ethylester (2.5 g, prepared by the procedure of Hercouet TetrahedronAsymmetry 1996, 7, 1267-1268.) and 7 M ammonia in MeOH (20 ml) washeated in a sealed vessel at 80° C. for 5 days. The cooled RM wasevaporated and triturated with hexanes/DCM to give the title compound asa beige solid. ¹H-NMR (DMSO-d₆, 400 MHz): 7.15 (s, 1H), 7.04 (s, 1H),3.00-2.91 (m, 1H), 2.67 (q, 1H), 1.94-1.83 (m, 1H), 1.74-1.67 (m, 1H),1.64-1.55 (m, 1H), 1.38-1.31 (m, 1H), 0.90 (t, 1H)).

Intermediate P: (2S,3R)-3-Methyl-pyrrolidine-2-carboxylic acid amide

(2S,3R)-3-Methyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylic acidamide (Stage P.1, 1.1 g, 4.76 mmol) and Pd on charcoal 10% (0.101 g,0.947 mmol) in MeOH (20 mL) was shaken under a H₂ atm. for 46 h at rt.The RM was then filtered through a Fluoropore Membrane Filter (0.2 μmFG) and evaporated. The residue was dissolved in DCM and evaporated todryness to give the title compound as white crystals MS: M+H=129.0.¹H-NMR (d₆-DMSO, 600 MHz): 7.34 (s, br,1H), 7.10 (s, br,1H), 3.48 (d,1H), 3.0.2-2.97 (m, 1H), 2.80-2.75 (m, 1H), 2.35-2.28 (m, 1H), 1.88-1.81(m, 1H), 1.39-1.32 (m, 1H), 0.83 (d, 3H).

Stage P.1:(2S,3R)-3-Methyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylic acidamide

Trimethylaluminum in toluene (2 M, 3.23 mL) was added dropwise to amixture of ammonium chloride (0.346 g, 6.47 mmol) in toluene (3.2 mL) at0° C. under an argon atm., with the formation of methane gas. The RM wasthen allowed to warm to rt, and stirred at rt for a further 15 min.before (2S,3R)-3-methyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester (prepared as described in Tetr. Lett. 1997, 38 (1),85-88; 1.6 g, 6.47 mmol) was slowly added. The RM was stirred at it for56 h, 1 M HCl was then added with cooling and the RM washed 3× with DCM.The aqueous phase was basified with Na₂CO₃, extracted 3× with DCM andthe combined organic layers dried over Na₂SO₄. Evaporation gave thetitle compound as a yellowish oil. MS: M+H=233.2. HPLC: t_(R) 3.17 min(method D). ¹H-NMR (d₆-DMSO, 600 MHz): 7.37-7.33 (m, 2H), 7.30 (t, 2H),7.25 (s, br,1H), 7.21 (t, 1H), 7.12 (s, br,1H), 3.55 (q, 1H), 3.40 (d,1H), 2.71 (t, 1H), 2.28-2.22 (m, 1H), 2.21-2.16 (m, 1H), 1.71 (qt, 1H),1.38-1.31 (m, 1H), 1.21 (d, 3H), 0.90 (d, 3H).

Intermediate Q: (2S,4S)-4-Dimethylamino-pyrrolidine-2-carboxylic acidamide

A solution of (2S,4S)-4-dimethylamino-pyrrolidine-2-carboxylic acidbutyl ester (Stage Q.1, 326 mg) and 7 M ammonia in MeOH (8 ml) was stoodfor 18 h at it in a sealed vessel. Filtration, evaporation andtrituration with Et₂O/MeOH gave the title compound as a beige solid.

Stage Q.1: (2S,4S)-4-Dimethylamino-pyrrolidine-2-carboxylic acid butylester

Concentrated HCl (0.3 ml) was added to a mixture of(2S,4S)-4-dimethylamino-pyrrolidine-2-carboxylic acid methyl esterdihydrochloride (400 mg) and 1-butanol (4 ml) and heated for 18 h at115° C. After cooling the RM was evaporated then partitioned between DCMand aqueous NaHCO₃ solution and the DCM layers dried and evaporated togive the title compound as a brown oil which was used without furtherpurification.

Intermediate R: (2S,4S)-4-Hydroxy-pyrrolidine-2-carboxylic acid amide

A solution of (2S,4S)-4-hydroxy-pyrrolidine-2-carboxylic acid methylester hydrochloride (1 g) in a 7M solution of ammonia in MeOH (10 ml)was stirred for 18 h then evaporated and triturated with Et₂O. Theresidue was dissolved in the minimum volume of hot MeOH and stood at 4°C. for 4 h. The title compound was isolated by filtration as a whitesolid.

Intermediate S: (2S,4R)-4-hydroxy-pyrrolidine-2-carboxylic acid amide

A solution of (2S,4R)-4-hydroxy-pyrrolidine-2-carboxylic acid benzylester (1 g) in 880 ammonia (5 ml) was stirred for 18 h then evaporatedand triturated with Et₂O to give the title compound as a white solid.¹H-NMR (d₆-DMSO, 400 MHz): 9.15 (s, br, 1H), 8.04 (s, 1H), 7.63 (s, 1H),5.56 (s, 1H), 4.40 (s, 1H), 4.27-4.16 (m, 1H), 3.27 (d, 1H), 3.02 (d,1H), 2.33 -2.19 (m, 1H), 1.89-1.76 (m, 1H).

Intermediate T: Imidazole-1-carboxylic acid(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide

7-tert-Butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-ylamine (StageT.1, 175 mg, 0.659 mmol) was dissolved in DCM (10 mL), CDI (297 mg,1.648 mmol) was then added, and the RM was stirred at rt for 2 h. Thetitle compound was isolated by filtration, washed with DCM and driedunder HV.

Stage T.1:7-tert-Butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-ylamine

N-(7-tert-Butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-acetamide(Stage T.2, 225 mg, 0.732 mmol) was dissolved in EtOH (10 mL), HCl 36%(1.48 g, 14.64 mmol) was then added, and the RM heated to reflux. After18 hours at reflux the RM was cooled to rt and adjusted to pH 8-9 by theaddition of 5% aqueous sodium bicarbonate solution, extracted withEtOAc, and washed two times with H₂O. The organic layer was dried overNa₂SO₄ and evaporated to give the title product (t_(R) 4.408 min (MethodF)).

Stage T.2:N-(7-tert-Butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-acetamide

N-(6-Bromo-7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide (StageT.3, 473 mg, 1.635 mmol) was dissolved in MeOH (10 mL), 2,2-dimethylthiopropionamide (230 mg, 1.962 mmol) and ammonium phosphomolybdate (307 mg,0.164 mmol) were added, and the RM was stirred at 25° C. for 20 h. TheRM was then stood for 2 days before stirring at 50° C. for 24 h. Themixture was extracted with EtOAc/H₂O. The organic layers were dried overNa₂SO₄ and evaporated. The crude material was chromatographed with 30 gof silica gel, eluent DCM/MeOH=99:1. Fractions containing the productwere evaporated and lyophilized from dioxane to give 225 mg of the titlecompound as a white solid (M+H=308; M−H=306; t_(R) 5.525 min (MethodF)).

Stage T.3:N-(6-Bromo-7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide

N-(7-Oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide (Stage T.4,2.286 g, 10.87 mmol) was dissolved in AcOH (60 ml), then bromine (1.74g, 10.87 mmol) dissolved in AcOH (10 mL) were slowly added and the RMwas heated to 75° C. for 20 h. The color changed from red to beige. Themixture was evaporated and the residue was dissolved in MeOH (10 mL) andprecipitated with H₂O. The mixture was filtered and dried on HV. Thecrude material was chromatographed with MPLC C18 H₂O 0.1% TFA/CH₃CN 0.1%TFA, gradient 0-50%. Fractions containing the product were neutralizedwith NaHCO₃, extracted with EtOAc and lyophilized from dioxane to giveof the title compound as a white solid (M+H=291; M−H=289; t_(R) 4.29(Method F)). ¹H-NMR (d₆-DMSO, 600.13 MHz) 12.75 (s, 1H) 4.95 (t, 1H),2.95-2.84 (m, 2H), 2.62-2.52 (m, 1H), 2.40-2.30 (m, 1H), 2.20 (s, 3H).

Stage T.4: N-(7-Oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide

To acetic anhydride (80 mL) was added at rt2-amino-5,6-dihydro-4H-benzothiazol-7-one (10 g, 59.4 mmol) and theyellow suspension was heated to reflux. After 1.75 h stirring at thistemperature, the RM was allowed to cool with stirring, and stirredovernight at rt. After further cooling with an ice/NaCl bath, thesuspension was filtered. The solid was then refluxed twice with acetone(10 mL then 15 mL) and filtered. The resulting solid was dried undervacuum at 40° C. overnight to afford the title compound as a beige solid(HPLC: t_(R) 3.47 min (Method A3), M−H=211.1), ¹H-NMR in DMSO-d6 (600MHz): 12.55 (s, br, 1H); 2.84 (t, 2H); 2.48 (t, 2H); 2.17 (s, 3H); 2.065(qt, 2H)).

Intermediate U: Imidazole-1-carboxylic acid[7-(2-fluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl]-amide

7-(2-Fluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-lamine(Stage U.1 61.6 mg, 0.217 mmol) was dissolved in DCM (2 mL), and CDI(297 mg, 1.648 mmol) added to give a clear colorless solution. The RMstood overnight at rt to give a white suspension. The mixture was cooledat 4° C. for 1 h and then filtered, washed with DCM and dried undervacuum to give of the title compound as a white solid (analysis of asample in MeOH; M+H=342.0 showed methyl carbamate product in MS; t_(R)2.14 min (Method A3)).

Stage U.17-(2-Fluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-lamine

3-Fluoro-2,2-dimethyl-thiopropionamide (Stage U.2, 394 mg, 2.331 mmol)was dissolved in EtOH,N-(6-bromo-7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide (StageT.3, 473 mg, 1.635 mmol) and ammonium phosphomolybdate (80 mg, 0.042mmol) were then added to give a yellow suspension. The RM was heated toreflux to give a dark blue-green suspension. The reaction mixture wasstirred at 65° C. 3 days. The RM was filtered and the filtrateevaporated. The residue was taken up in DMF and purified by prep-HPLC(method E). Fractions containing the product were combined andevaporated to give the title compound as a white solid (M+H=284.1; t_(R)1.30 min (Method A3)).

Stage U.2 3-Fluoro-2,2-dimethyl-thiopropionamide

The title compound was prepared by the procedure of Boys, M. L.; Downs,V. L. Synth. Commun. 2006, 36, 295. Sodium hydrogen sulphide hydrate(3.89 g, 69.4 mmol, hygroscopic) was added to a solution of3-fluoro-2,2-dimethyl-propionitrile (Stage U.3, 1.17 g, 11.57 mmol) anddiethyl amine hydrochloride (7.61 g, 69.4 mmol) in 1,4-dioxane (7 mL)and H₂O (7 mL) at rt. The RM was heated to 55° C. and then stirred for 3days at this temperature. The reaction mixture was diluted with water(50 ml) and extracted 5× with EtOAc (50 mL). The organic layers weredried over Na₂SO₄ and evaporated to give an orange oil. DCM (5 mL) wasthen added to give a white suspension. The suspension was filtered andthe filtrate was purified with flash chromatography on silica geleluting with DCM. The product containing fractions were evaporated togive the title compound as a pale yellow oil (M+H=136.1, M−H=134.1;t_(R) 0.69 min (Method A3)). ¹⁹F-NMR (d₆-DMSO, 400 MHz 218 ppm (t, 1F)).

Stage U.3 3-Fluoro-2,2-dimethyl-propionitrile

3-Fluoro-2,2-dimethyl-propionamide (Stage U.4, 1.82 g, 15.28 mmol) andphosphorus pentoxide (2.168 g, 15.28 mmol) were combined to give a freeflowing white powder which was heated with an oil bath to a bathtemperature of 180° C. over 50 minutes under an argon atmosphere. A 300mbar vacuum was then slowly applied distilling a mobile clear colourlessoil which formed a low melting waxy solid on standing of the titlecompound. ¹⁹F-NMR (d₆-DMSO, 400 MHz 219.5 ppm (t, 1F)).

Stage U.4 3-Fluoro-2,2-dimethyl-propionamide

3-Fluoro-2,2-dimethyl-propionyl fluoride (DE3326874 and DE3611195, 2.5g, 20.47 mmol) was added at 0° C. to a mixture of aqueous ammonia (10mL) and THF (20 mL). The RM was then allowed to warm to rt and stoodovernight at rt. The volume was reduced by 50% under vacuum to give athick white suspension. The suspension was extracted with DCM/H₂O. Theorganic layers were dried over Na₂SO₄ and evaporated to give the titlecompound as a white crystalline solid.

Intermediate V: Imidazole-1-carboxylic acid(7-cyclopropylmethyl-4,5-dihydro-benzo[1,2-;3,4-d′]bisthiazol-2-yl)-amide

7-Cyclopropylmethyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-ylamine(Stage V.1, 72 mg, 0.273 mmol) was dissolved in 5 mL DCM, CDI (73.9 mg,0.410 mmol) was added, and the RM stirred at rt for 20 h. Additional CDI(37 mg, 0.205 mmol) was added and the RM stirred at rt for a further 2hours. The mixture was filtered, washed with DCM and filtrate wasevaporated to give the title compound (M+H=322.1; M−H=320.2 MS-ES inMeOH shows the methyl carbamate product).

Stage V.17-Cyclopropylmethyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-ylamine

N-(7-Cyclopropylmethyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-acetamide(Stage V.2, 180 mg, 0.589 mmol) was dissolved in EtOH (10 mL) and 36%HCl (1.193 g, 11.79 mmol) added. The RM was heated to 90° C. for 16 hthen cooled and extracted with EtOAc/H₂O. The organic layer was driedover Na₂SO₄ and evaporated. The crude material was chromatographed withMPLC C18 H₂O 0.1% TFA/CH₃CN 0.1% TFA, gradient 0-50%. Product containingfractions were neutralized with NaHCO₃, extracted with EtOAc andlyophilized from dioxane to give the title compound (M+H=264.2;M−H=262.1; t_(R) 4.183 min (Method F)).

Stage V.2N-(7-Cyclopropylmethyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-acetamide

N-(6-Bromo-7-oxo-4,5,6,7-tetrahydro-benzothiazol-2-yl)-acetamide (StageT.3, 347 mg, 1.200 mmol) was dissolved in MeOH (10 mL) and2-cyclopropyl-thioacetamide (Can. J. Chem 1995 Vol. 73 1468-1477, 166mg, 1.440 mmol) and ammonium phosphomolybdate (225 mg, 0.120 mmol) wereadded. The RM was stirred at rt for 20 h then heated at 50° C. for 3 h,60° C. for 2 h and then refluxed for 24 h. The RM was partitionedbetween EtOAc/H₂O. The organic layer was dried over Na₂SO₄ andevaporated. The raw material was chromatographied with MPLC C18 H₂O 0.1%TFA/CH₃CN 0.1% TFA, gradient 0-50%. Product containing fractions wereneutralized with NaHCO₃, extracted with EtOAc and lyophilized fromdioxane to give the title compound as a white solid (M+H=306.2;M−H=304.2; t_(R) 5.12 min (Method F)).

EXAMPLE 1 (2S,4R)-4-Dimethylamino-pyrrolidine-1,2-dicarboxylic acid2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To a solution of imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 45.1 mg, 0.127 mmol) in DMF (1 mL) was added(2S,4R)-4-dimethylamino-pyrrolidine-2-carboxylic acid amide(Intermediate B, 22 mg, 0.140 mmol) and triethylamine (0.053 ml, 0.382mmol). The RM was then stirred at rt for 17 h and purified bypreparative HPLC (method B). The product containing fractions werecombined and eluted through a Bond Elut-SCX, 300 mg cartridge. Thecartridge was then washed with a 7 M solution of ammonia in MeOH andevaporated to give the title compound. MS: M+H=444. HPLC: t_(R) 3.17 min(method D).

EXAMPLE 2 (2S,3S)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

A mixture of (2S,3S)-3-methyl-pyrrolidine-2-carboxylic acid amide(Intermediate D, 6.24 mg, 0.049 mmol), imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 18 mg, 0.049 mmol) and triethylamine (0.020 mL, 0.146mmol) in DMF (0.4 mL) was stirred at rt for 6 h. The RM was thendirectly purified by preparative HPLC (method B). The product containingfractions were then filtered through a 300 mg Bond Elut-SCX cartridge.The cartridge was then eluted with a 7 M ammonia solution in MeOH andthe eluent evaporated to give the title compound as a yellow solid. MS:M+H=430.1. HPLC: t_(R) 3.72 min (method D). ¹H-NMR (DMSO-d₆, 600 MHz):11.05 (s, 1H); 8.04 (s, 1H); 7.44 (s, br, 1H); 6.95 (s, br, 1H); 3.78(s, br, 1H); 3.60-3.50 (m, 6H); 2.82 (s, br, 4H); 2.18 (s, br, 1H); 2.03(s, br, 1H); 1.55 (s, br, 1H); 1.10 (t, 6H); 1.05 (d, 3H).

EXAMPLE 3 (R)-2-Benzyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To a mixture of imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 50 mg, 0.135 mmol) in DMF (1 mL) was added(R)-2-benzyl-pyrrolidine-2-carboxylic acid amide (Intermediate E, 33.2mg, 0.162 mmol) and triethylamine (0.057 mL, 0.406 mmol). The RM wasstirred for 21 h at 40° C. and then directly purified with preparativeHPLC (method B). The product containing fractions were eluted through a300 mg Bond Elut-SCX, cartridge. The cartridge was then eluted with 7 Mammonia-solution in MeOH and evaporated give the title compound as ayellow solid. MS: M+H=506.1. HPLC: t_(R) 4.38 min (method D).

EXAMPLE 4 (S)-2-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To a mixture of imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 75 mg, 0.203 mmol) in DMF (2 mL) was added(S)-2-methyl-pyrrolidine-2-carboxylic acid amide (Intermediate F, 39 mg,0.305 mmol) and triethylamine (0.085 mL, 0.609 mmol). The RM was stirredfor at 40° C. 17 h and then directly purified with preparative HPLC(method B). The product containing fractions were eluted through a 300mg Bond Elut-SCX cartridge. The cartridge was then eluted with 7 Mammonia-solution in MeOH and evaporated to give the title compound as ayellow solid MS: M+H=430.2. HPLC: t_(R) 3.80 min (method D).

EXAMPLE 5 (R)-2-Methoxymethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

A mixture of imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 75 mg, 0.203 mmol),(R)-2-methoxymethyl-pyrrolidine-2-carboxylic acid amide (Intermediate G,35.3 mg, 0.223 mmol) and triethylamine (0.085 mL, 0.609 mmol) in DMF (2mL) was stirred for 6 h at 40° C. After cooling to rt, the RM wasdissolved in MeOH (1 mL) and directly purified by preparative HPLC(method B). The product containing fractions were eluted through a 300mg Bond Elut-SCX cartridge. The cartridge was then eluted with 7 Mammonia solution in MeOH and evaporated to give the title product as ayellow solid. MS: M+H=460.1. HPLC: t_(R) 3.94 min (method D).

EXAMPLE 6 (R)-2-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylic acid2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

Imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 132 mg, 0.372 mmol) was added to a mixture of(R)-2-dimethylaminomethyl-pyrrolidine-2-carboxylic acid amide(Intermediate H, 70 mg, 0.409 mmol) and triethylamine (0.155 mL, 1.115mmol) in DMF (1 mL) at rt. After 18 hours MeOH (0.5 ml) was added, theRM filtered through a PTFE membrane filter and purified by preparativeHPLC (method B). The product containing fractions were combined andevaporated to remove CH₃CN and then basified by the addition of solidNaHCO₃ to give a yellow white precipitate. After cooling to 4° C., thesolid was recollected by filtration and then further purified bypreparative HPLC (method C). The product containing fractions wereeluted through a 300 mg Bond Elut-SCX cartridge. The cartridge was theneluted with a 7 M ammonia in MeOH solution and evaporated to give thetitle product as a yellow amorphous glass. LCMS: t_(R) 0.98 min andM+H=458.1 (method A3). ¹H-NMR (CD₃OD, 400 MHz): 8.28 (s, 1H); 3.92-3.82(m, 1H); 3.67-3.44 (m, 2H); 3.04-2.88 (m, 4H); 2.85-2.71 (m, 1H); 2.56(s, br, 6H); 2.29-2.19 (m, 1H); 2.08-1.87 (m, 3H); 1.37 (s, 9H).

EXAMPLE 7 d₆-(R)-2-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylic acid2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

Imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 91 mg, 0.256 mmol) and triethylamine (0.036 mL, 0.256mmol) were added tod₆-(R)-2-dimethylaminomethyl-pyrrolidine-2-carboxylic acid amide(Intermediate I, 50 mg, 0.282 mmol) suspended in DMF (2 mL) under anargon atm. The RM was then stirred for 2.75 h at 40° C. and thenpurified directly by preparative HPLC twice (method B and then methodC). Each time, fractions containing the product were combined and elutedthrough a 300 mg Bond Elut-SCX cartridge. The cartridge was then elutedwith a 7 M ammonia in MeOH solution and evaporated to give a yellowsolid. The solid was suspended in DCM, filtered and dried in vacuo togive the title compound as a yellow solid. LCMS: t_(R) 1.21 min andM+H=464.0 (method A1).

EXAMPLE 8 (R)-2-Hydroxymethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

Imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 50 mg, 0.135 mmol) was added to a mixture of(R)-2-hydroxymethyl-pyrrolidine-2-carboxylic acid amide (Intermediate J,25.4 mg, 0.176 mmol) and triethylamine (0.047 mL, 0.338 mmol) in DMF (1mL) at rt. After 18 h at rt the RM was filtered through a PTFE membraneand purified by preparative HPLC (method B). Product containingfractions the were combined and filtered through a Bond Elut-SCXcartridge. The cartridge was then eluted with a 7 M ammonia in MeOHsolution and evaporated to give an orange glass which was recrystallizedfrom MeOH/water to afford the title compound as a yellow solid. LCMS:t_(R) 1.09 min and M+H=446.0 (method A3).

EXAMPLE 9 (R)-2-Hydroxymethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

Imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 124 mg, 0.350 mmol) was added to a mixture of(R)-2-hydroxymethyl-pyrrolidine-2-carboxylic acid amide (Intermediate J,63 mg, 0.350 mmol) and triethylamine (0.049 mL, 0.350 mmol) in DMF (2mL). The RM was stirred for 2 h at 40° C. then stood for 18 h at rt andthen directly purified by preparative HPLC (method B). The productcontaining fractions were eluted through a 300 mg Bond Elut-SCXcartridge. The cartridge was then eluted with 7 M solution of ammonia inMeOH and evaporated to give a yellow solid. The isolated solid was thensuspended in DCM, filtered and dried to give title compound as a paleyellow/white solid. LCMS: t_(R) 1.31 min and M+H=430.9 (method A1).

EXAMPLE 10(R)-2-{[(3-Fluoro-benzyl)-methyl-amino]-methyl}-pyrrolidine-1,2-dicarboxylicacid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

Imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 40.1 mg, 0.113 mmol) and triethylamine (0.047 mL, 0.339mmol) were added to(R)-2-{[(3-fluoro-benzyl)-methyl-amino]-methyl}-pyrrolidine-2-carboxylicacid amide (Intermediate K, 30 mg, 0.113 mmol) suspended in DMF (1 mL)at rt under argon. The RM was stirred for 2.5 h at 40° C. and thendirectly purified by preparative HPLC (method C). The product containingfractions were eluted through a Bond Elut-SCX, 300 mg cartridge. Thecartridge was then eluted with a 7 M solution of ammonia in MeOH andevaporated to give a yellow solid. The solid was triturated with DCM,filtered and dried to give the title compound as a pale yellow solid.LCMS: t_(R) 0.81 min and M+H=552.3 (method A2).

EXAMPLE 11 (S)-2-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

(S)-2-Methyl-pyrrolidine-2-carboxylic acid amide (Intermediate F, 22.14mg, 0.169 mmol) and triethylamine (0.035 mL, 0.254 mmol) were added toimidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 30 mg, 0.085 mmol) in DMF (1 mL) under argon at rt.After stirring 16 h at rt the RM was directly purified by preparativeHPLC (method B). The product containing fractions were eluted through a300 mg Bond Elut-SCX cartridge. The cartridge was then eluted with a 7 Msolution of ammonia in MeOH and evaporated to give the title compound asa yellow crystalline solid. MS: M+H=415.1. HPLC: t_(R) 3.73 min (methodD).

EXAMPLE 12 (S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

(S)-Pyrrolidine-2-carboxylic acid amide (148 mg, 1.299 mmol) andtriethylamine (0.272 mL, 1.949 mmol) were added toimidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 240 mg, 0.65 mmol) and (DMF (1 mL) under argon at rt.The RM was stirred for 15.5 h at rt and then purified with preparativeHPLC (method C). Product containing fractions were combined and filteredthrough a Bond Elut-SCX cartridge. The cartridge was then eluted with a7 M solution of ammonia in MeOH and evaporated to give the titlecompound as orange crystals. MS: M+H=416.1. HPLC: t_(R) 3.56 min (methodD).

EXAMPLE 13 (S)-Azetidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

A mixture of (S)-azetidine-2-carboxylic acid amide (Intermediate L, 26.4mg, 0.264 mmol), imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 75 mg, 0.203 mmol) and triethylamine (0.085 mL, 0.609mmol) in DMF (2 mL) was stirred at 40° C. for 2 h. The RM was thenpurified directly by preparative HPLC (method B). The product containingfractions were eluted through a 300 mg Bond Elut-SCX cartridge. Thecartridge was then eluted with a 7 M solution of ammonia in MeOH andevaporated to give the title compound as a white crystalline solid. MS:M+H=402.1. HPLC: t_(R) 3.56 min (method D).

EXAMPLE 14 (S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

(S)-Pyrrolidine-2-carboxylic acid amide (16 mg, 0.14 mmol) was added toa mixture of imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 45 mg, 0.127 mmol) and triethylamine (0.053 mL, 0.381mmol) in DMF (1 mL) at rt. The RM was stood overnight at rt, thenevaporated and the residue crystallized from MeOH and water. The titlecompound was collected by filtration. MS: M+H=401.1 (MS-ESI). ¹H-NMR(CD₃OD, 400 MHz): 8.31 (s, 1H), 4.46 (d, 1H), 3.77-3.54 (m, 2H),3.08-2.90 (m, 4H), 2.33-2.23 (m, 1H), 2.13-2.00 (m, 3H), 1.38 (s, 9H)).

EXAMPLE 15 5-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To a suspension of imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 75 mg, 0.203 mmol) in DMF (2 mL) was added5-methyl-pyrrolidine-2-carboxylic acid amide (prepared as described inArch. Pharm., 1936, 274, 40; 39 mg, 0.305 mmol) and triethylamine (0.085mL, 0.609 mmol). The RM was stirred at 40° C. for 2.5 h. and thenpurified directly by preparative HPLC (method B). The product containingfractions were eluted through a Bond Elut-SCX cartridge. The cartridgewas then eluted with a 7 M solution of ammonia in MeOH, evaporated andthe residue triturated with DCM to give the title compound as a yellowsolid. LCMS: t_(R) 0.77 min and M+H=430.0 (method A2).

EXAMPLE 16 (2S,4R)-4-Fluoro-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 75 mg, 0.203 mmol) in DCM (2 mL) was added(2S,4R)-4-fluoro-pyrrolidine-2-carboxylic acid amide (Intermediate M,40.2 mg, 0.305 mmol) and triethylamine (0.085 mL, 0.609 mmol). The RMwas stirred at 40° C. for 17.5 h and then directly purified bypreparative HPLC (method B). The product containing fractions wereeluted through a Bond Elut-SCX cartridge. The cartridge was then elutedwith a 7 M solution of ammonia in MeOH and evaporated to give the titlecompound as a yellow solid. HPLC: t_(R) 3.66 min (method D). MS:M+H=434.1.

EXAMPLE 17 (2S,4S)-4-Fluoro-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 17 mg, 0.046 mmol) in DMF (0.4 mL) was added(2S,4S)-4-fluoro-pyrrolidine-2-carboxylic acid amide (Intermediate N,6.08 mg, 0.046 mmol) and triethylamine (0.019 mL, 0.138 mmol). The RMwas stirred at 40° C. for 17 h and then directly purified by preparativeHPLC (method B). The product containing fractions were eluted through aBond Elut-SCX cartridge. The cartridge was then eluted with a 7 Msolution of ammonia in MeOH and evaporated to give the title compound asa yellow solid. HPLC: t_(R) 3.55 min (method D). MS: M+H=434.1.

EXAMPLE 18 (1S,5R)-2-Aza-bicyclo[3.1.0]hexane-1,2-dicarboxylic acid1-amide2-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

(1S,5R)-2-Aza-bicyclo[3.1.0]hexane-1-carboxylic acid amide (IntermediateO, 208 mg, 1.65 mmol) was added to a stirred mixture ofimidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 532 mg, 1.5 mmol) and triethylamine (0.627 mL, 4.50mmol) in DMF (4 mL) at rt. After 56 and 80 hours additional portions of(1S,5R)-2-aza-bicyclo[3.1.0]hexane-1-carboxylic acid amide (IntermediateO, 104 mg, 0.825 mmol) were added and the RM stood 18 h at rt. The RMwas then filtered through a PTFE membrane and purified by preparativeHPLC (method B). The product containing fractions were eluted through aBond Elut-SCX cartridge. The cartridge was then eluted with 7 Mammonia-solution in MeOH and evaporated. The residue was purified for asecond time by preparative HPLC (method B) and isolated in the samemanner. The crude product was then recrystallised from a 1:1 mixtre ofwater/MeOH to give the title compound as a yellow crystalline solid.LCMS: t_(R) 1.22 min and M+H=413.1 (method A3). ¹H-NMR (d₆-DMSO, 400MHz): 11.22 (s, 1H), 8.40 (s, 1H); 7.33 (s, br, 1H); 7.05 (s, br, 1H);3.97-3.84 (m, 1H); 3.64-3.52 (m, 1H); 3.01-2.83 (m, 4H); 2.28-2.13 (m,1H); 1.95-1.74 (m, 3H); 1.33 (s, 9H); 1.04-0.97 (m, 1H)).

EXAMPLE 19 (1S,5R)-2-Aza-bicyclo[3.1.0]hexane-1,2-dicarboxylic acid1-amide2-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 75 mg, 0.203 mmol) in DCM (2 mL) was added(1S,5R)-2-aza-bicyclo[3.1.0]hexane-1-carboxylic acid amide (IntermediateO, 38.4 mg, 0.305 mmol) and triethylamine (0.085 mL, 0.609 mmol). The RMwas stirred at 40° C. for 17.5 h and then directly purified bypreparative HPLC (method B). The product containing fractions wereeluted through a Bond Elut-SCX cartridge. The cartridge was then elutedwith a 7 M solution of ammonia in MeOH and evaporated to give the titlecompound as a yellow solid. HPLC: t_(R) 3.73 min (method D). MS:M+H=428.1.

EXAMPLE 20 (2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 60 mg, 0.169 mmol) in DMF (1 mL) was added(2S,3R)-3-methyl-pyrrolidine-2-carboxylic acid amide (Intermediate P,32.5 mg, 0.254 mmol) and triethylamine (0.071 mL, 0.508 mmol). The RMwas stirred under argon for 2 h at rt and then directly purified bypreparative HPLC (method B). The CH₃CN was evaporated from the productcontaining fractions and the remaining liquid then eluted through a BondElut-SCX cartridge. The cartridge was then eluted with a 7 M solution ofammonia in MeOH and evaporated. The residue was triturated in methanoland the title compound collected as white crystals following filtrationand drying. HPLC: t_(R) 3.66 (method D). MS: M+H=415.1. ¹H-NMR (CD₃OD,600 MHz): 7.96 (s, 1H), 4.04 (d, 1H), 3.47 (t, 1H), 3.18 (q, 1H),2.71-2.67 (m, 2H), 2.64-2.58 (m, 2H), 2.24-2.15 (m, 1H), 1.76 (s, br,1H), 1.53 (s, br, 1H), 1.03 (s, 9H), 0.78 (d, 3H).

EXAMPLE 21 (2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 82 mg, 0.222 mmol) in DMF (1.5 mL) was added underargon (2S,3R)-3-methyl-pyrrolidine-2-carboxylic acid amide (IntermediateP, 42.7 mg, 0.333 mmol) and triethylamine (0.093 mL, 0.666 mmol). The RMwas stirred at rt for 2 h and then directly purified by preparative HPLC(method B). The CH₃CN was removed under vacuum from the productcontaining fractions and the remaining solution then eluted through aBond Elut-SCX cartridge. The cartridge was then eluted with a 7 Mammonia solution in MeOH, evaporated and the residue triturated withmethanol to give the title compound as a white crystalaline solid. HPLC:t_(R) 3.74 (method D). MS: M+H=430.1. ¹H-NMR (CD₃OD, 600 MHz): 7.62 (s,1H), 4.03 (d, 1H), 3.46 (t, 1H), 3.26 (q, 4H), 3.17 (q, 1H), 2.54 (s,4H), 2.24-2.15 (m, 1H), 1.80-1.72 (m, 1H), 1.58-1.47 (m, 1H), 0.83 (t,6H), 0.77 (d, 3H).

EXAMPLE 22 (2S,4S)-4-Dimethylamino-pyrrolidine-1,2-dicarboxylic acid2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 60 mg, 0.162 mmol) in DMF (1.5 mL) was added(2S,4S)-4-dimethylamino-pyrrolidine-2-carboxylic acid amide(Intermediate Q, 28.1 mg, 0.179 mmol) and triethylamine (0.068 mL, 0.487mmol). The RM was stirred at 40° C. for 17 h and then directly purifiedby preparative HPLC (method B). The product containing fractions werepassed through a Bond Elut-SCX cartridge. The cartridge was then elutedwith a 7 M ammonia solution in MeOH. The solution was evaporated to givethe title compound. HPLC: t_(R) 3.23 min (method D). MS: M+H=459.1.

EXAMPLE 23 5-Phenyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To a suspension of imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 65 mg, 0.176 mmol) in DMF (2 mL) were added5-phenyl-pyrrolidine-2-carboxylic acid amide (prepared as described inpatent U.S. Pat. No. 3,164,597, Example 42, 50.2 mg, 0.264 mmol) andtriethylamine (0.074 mL, 0.528 mmol). The RM was stirred at 40° C. for 2h and then directly purified by preparative HPLC (method B). The productcontaining fractions were passed through a Bond Elut-SCX cartridge. Thecartridge was then eluted with a 7 M ammonia solution in MeOH. Thesolution was evaporated and the residue was triturated in DCM to givethe title compound as a yellow solid. LCMS: t_(R) 0.99 min and M+H=492.0(method A2).

EXAMPLE 24 Azetidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To a solution of imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 100 mg, 0.282 mmol) in DMF (2 mL) were addedazetidine-2-carboxylic acid amide (56.5 mg, 0.564 mmol) andtriethylamine (0.118 mL, 0.846 mmol). The RM was stirred under argonatm. at rt for 16 h and then directly purified by preparative HPLC(method B). The product containing fractions were passed through a BondElut-SCX cartridge. The cartridge was then eluted with a 7 M ammoniasolution in MeOH. The solution was evaporated and the residue purifiedby flash chromatography on silica gel, eluting with DCM/MeOH 95:5containing 0.5% 880 HN₃ to give the title compound as a yellow solid.HPLC: t_(R) 3.55 min (method D). MS: M+H=387.1.

EXAMPLE 25 (S)-Azetidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

(S)-Azetidine-2-carboxylic acid amide (Intermediate L, 68.1 mg, 0.680mmol) was added to a stirred mixture of imidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A, 241 mg, 0.680 mmol) and triethylamine (0.284 mL, 2.040mmol) in DMF (3 mL) at rt. The RM was allowed to stand 66 h at rt andthen directly purified by preparative HPLC (method B). The productcontaining fractions were evaporated to remove the CH₃CN and thenbasified with NaHCO₃. The aqueous phase was then extracted 4× with 10%MeOH in DCM, the combined organic layers dried over Na₂SO₄ andevaporated to give a solid which was triturated with MeOH/water to givethe title compound as an off-white crystaline solid. LCMS: t_(R) 1.08min and M+H=386.9 (method A3).

EXAMPLE 26 (2S,4S)-4-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To a suspension of imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 75 mg, 0.203 mmol) in DMF (2 mL) were added(2S,4S)-4-hydroxy-pyrrolidine-2-carboxylic acid amide (Intermediate R,39.6 mg, 0.305 mmol) and triethylamine (0.113 mL, 0.812 mmol). The RMwas stirred at 40° C. for 2.5 h and then directly purified bypreparative HPLC (method B). The product containing fractions werepassed through a Bond Elut-SCX cartridge. The cartridge was then elutedwith a 7 M ammonia solution in MeOH. The solution was evaporated to givethe title compound as a yellow solid. HPLC: t_(R) 3.44 min (method D).MS: M+H=432.1.

EXAMPLE 27 (2S,4R)-4-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

To a suspension of imidazole-1-carboxylic acid(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate C, 75 mg, 0.203 mmol) in DMF (2 mL) were added(2S,4R)-4-hydroxy-pyrrolidine-2-carboxylic acid amide (Intermediate S,39.6 mg, 0.305 mmol) and triethylamine (0.113 mL, 0.812 mmol). The RMwas stirred at 40° C. for 4.5 h and then directly purified bypreparative HPLC (method B). The product containing fractions werepassed through a Bond Elut-SCX cartridge. The cartridge was then elutedwith a 7 M ammonia solution in MeOH. The solution was evaporated and theresidue was recrystallised from DCM to give the title compound as ayellow solid. HPLC: t_(R) 3.31 min (method D). MS: M+H=432.1.

EXAMPLE 28 (S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

L-Prolinamide (59.4 mg, 0.521 mmol) and TEA (0.121 mL, 0.868 mmol) wereadded to imidazole-1-carboxylic acid(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide(Intermediate T, 156 mg, 0.434 mmol) in DMF (1 mL). The RM was stirredat rt for 10 min. The mixture was extracted with EtOAc/H₂O. The organiclayer was dried over Na2SO4 and evaporated. The crude material waschromatographied with MPLC C18 H₂O 0.1% TFA/CH₃CN 0.1% TFA, gradient0-50%. Product containing fractions were neutralized with NaHCO₃,extracted with EtOAc and lyophilized from dioxane to give the titlecompound as a white solid (M+H=406; M−H=404; t_(R) 4.75 min (Method F).¹H-NMR (d₆-DMSO, 600.13 MHz) 10.80 (s, 1H) 7.40 (s, 1H), 6.98 (s, 1H),4.30 (s, 1H) 3.65-3.55 (m 1H), 3.48-3.38 (m, 1H), 3.10 (t, 2H), 2.94 (t,2H), 2.14-2.05 (m, 1H), 1.95-1.78 (m, 3H), 1.40 (s, 9H)).

EXAMPLE 29 (S)-2-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

(S)-2-Methyl-pyrrolidine-2-carboxylic acid amide (Intermediate F, 7.7mg, 0.060 mmol) and TEA (0.014 mL, 0.100 mmol) were added toimidazole-1-carboxylic acid(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide(Intermediate T, 18 mg, 0.050 mmol) in DMF (3 mL). The RM was stirred atrt for 10 min. then extracted with EtOAc/H₂O. The organic layer wasdried over Na₂SO₄ and evaporated. The crude material waschromatographied with MPLC C18 H₂O 0.1% TFA/CH₃CN 0.1% TFA, gradient0-50%. Product containing fractions were neutralized with NaHCO₃,extracted with EtOAc and lyophilized from dioxane to give the titlecompound as a white solid (M+H=420; M−H=418; t_(R) 4.875 min (Method F).¹H-NMR (d₆-DMSO, 600.13 MHz) 10.60 (s, br, 1H) 7.68 (s, 1H), 6.86 (s,1H), 3.68-3.60 (m 1H), 3.60-3.50 (m 1H), 3.08 (t, 2H), 2.90 (t, 2H),2.10-1.98 (m, 1H), 1.90-1.68 (m, 3H), 1.48 (s, 3H), 1.37 (s, 9H)).

EXAMPLE 30 (S)-Azetidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

(S)-Azetidine-2-carboxylic acid amide (Intermediate L, 37.1 mg, 0.370mmol) and TEA (0.14 mL, 1.010 mmol) were added to imidazole-1-carboxylicacid (7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide(Intermediate T, 121 mg, 0.337 mmol) in DMF (2 mL). The RM was stirredfor 5 min and then stood for 2 days at rt. The RM was filtered anddirectly purified by Prep.HPLC (method E). Product containing fractionswere combined and filtered through a Bond Elute-SCX, 300 mg cartridge.The cartridge was then washed with 7 M ammonia-solution in MeOH (2 ml).The filtrate was evaporated to give the title compound as a white solid(M+H=391.8; M−H=389.8; t_(R) 1.84 min (Method A3). ¹H-NMR (d₆-DMSO, 400MHz) 11.12 (s, 1H) 7.52 (s, 1H), 7.30 (s, 1H), 4.74-4.62 (m, 1H), 3.92(t, 2H), 3.08 (t, 2H), 2.90 (t, 2H), 2.46-2.36 (m, 1H), 2.18-2.02 (m,1H), 1.39 (s, 9H)).

EXAMPLE 31 (2S,4R)-4-Dimethylamino-pyrrolidine-1,2-dicarboxylic acid2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

(2S,4R)-4-Dimethylamino-pyrrolidine-2-carboxylic acid amide(Intermediate B, 9.33 mg, 0.059 mmol) and TEA (0.019 ml, 0.135 mmol)were added to imidazole-1-carboxylic acid(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide(Intermediate T, 19.4 mg, 0.054 mmol) in DMF (1 ml). The RM was stirredfor 5 min and then stood overnight at rt. The RM was filtered anddirectly purified by Prep.HPLC (method E). Fractions containing theproduct were combined and filtered through a Bond Elute-SCX, 300 mgcartridge. The cartridge was then washed with 7 M ammonia-solution inMeOH. The filtrate was evaporated to give the title compound as a whitesolid (M+H=449.1; M−H=447.3; t_(R) 1.47 min (Method A3). ¹H-NMR(d₆-DMSO, 400 MHz) 7.40 (s, 1H), 7.00 (s, 1H), 4.44-4.25 (m, 1H),3.86-3.76 (m 1H), 3.35-3.25 (m 1H), 3.08 (t, 2H), 2.92 (t, 2H),2.96-2.78 (m, 1H), 1.18 (s, br, 6H), 2.08-1.85 (m, 2H), 1.38 (s, 9H)).

EXAMPLE 32 (S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[7-(2-fluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl]-amide}

L-Prolinamide (23.98 mg, 0.210 mmol) and TEA (0.080 mL, 0.573 mmol) wereadded to imidazole-1-carboxylic acid[7-(2-fluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl]-amide(Intermediate U, 72.1 mg, 0.191 mmol) in DMF (1 mL). The RM was stirredfor 5 min and then stood overnight at rt. The RM was evaporated and thentriturated with MeOH (2 mL) and H₂O (1 mL). The mixture was cooled to 4°C. The suspension was filtered and washed with cold MeOH/H₂O=2:1 and thesolid dried under HV at 40° C. to give the title compound as a whitesolid (M+H=424.0; M−H=422.1; t_(R) 1.7 min (Method A3). ¹H-NMR (d₆-DMSO,400 MHz) 7.19 (s, 1H), 6.96 (s, 1H), 4.58 (s, 1H), 4.44 (s, 1H), 4.28(s, br, 1H) 3.64-3.50 (m 1H), 3.48-3.38 (m 1H), 3.10 (t, 2H), 2.92 (t,2H), 2.18-1.98 (m, 1H), 1.94-1.78 (m, 3H), 1.38 (s, 6H) ¹⁹F-NMR(d6-DMSO, 600.13 MHz) 219 ppm (t, 1F)).

EXAMPLE 33 (S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-cyclopropylmethyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

L-Prolinamide (16.7 mg, 0.147 mmol) and TEA (0.027 mL, 0.196 mmol) wereadded to imidazole-1-carboxylic acid(7-cyclopropylmethyl-4,5-dihydro-benzo[1,2-;3,4-d′]bisthiazol-2-yl)-amide(Intermediate V, 35 mg, 0.098 mmol) in DMF (1.5 mL). The RM was stirredat rt for 15 min and then extracted with EtOAc/H₂O. The organic layerwas dried over Na₂SO₄ and evaporated. The crude material waschromatographied with MPLC C18 H₂O 0.1% TFA/CH₃CN 0.1% TFA, gradient0-50%. Product containing fractions were neutralized with NaHCO₃,extracted with EtOAc and lyophilized from dioxane to give the titlecompound as a white solid (M+H=404; M−H=402; t_(R) 4.49 min (Method F).¹H-NMR (d₆-DMSO, 600.13 MHz) 10.78 (s, 1H) 7.40 (s, 1H), 6.98 (s, 1H),4.28 (s, br, 1H) 3.65-3.55 (m, 1H), 3.48-3.38 (m, 1H), 3.08 (t, 2H),2.97 (t, 2H), 2.84 (d, 2H), 2.14-2.04 (m, 1H), 1.93-1.72 (m, 3H), 1.10(q, 1H), 0.60-0.50 (m, 2H), 0.40-0.38 (m, 2H)).

EXAMPLE 34 (2S,3S)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

(2S,3S)-3-Methyl-pyrrolidine-2-carboxylic acid amide (Intermediate D,13.37 mg, 0.104 mmol) and TEA (0.039 mL, 0.278 mmol) were added toimidazole-1-carboxylic acid(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide(Intermediate T, 25 mg, 0.070 mmol) in DMF (2 mL). The RM was stirred atrt for 10 min and then extracted with EtOAc/H₂O. The organic layer wasdried over Na₂SO₄ and evaporated. The crude material was lyophilizedfrom dioxane without further purification to give the title compound asa white solid (M+H=420.1; M−H=418.1; t_(R) 4.85 min (Method F). ¹H-NMR(d₆-DMSO, 600.13 MHz) 10.78 (s, br, 1H) 7.44 (s, br, 1H), 6.97 (s, br,1H), 3.90-3.70 (m, 1H) 3.62-3.42 (m, 2H), 3.08 (t, 2H), 2.94 (t, 2H),2.22-2.12 (m, 1H), 2.06-1.94 (m, 1H), 1.58-1.48 (m, 1H), 1.39 (s, 9H),1.04 (d, 3H)).

EXAMPLE 35 (1S,5R)-2-Aza-bicyclo[3.1.0]hexane-1,2-dicarboxylic acid1-amide2-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

The title compound was synthesized in a similar manner as described forExample 34 using intermediate O instead of intermediate D.

(M+H=418.0; M−H=416.1; t_(R) 4.90 min (Method F). ¹H-NMR (d₆-DMSO,600.13 MHz) 10.78 (s, br, 1H) 7.40 (s, br, 1H), 7.06 (s, br, 1H),3.92-3.82 (m, 1H) 3.62-3.52 (m, 1H), 3.08 (t, 2H), 2.94 (t, 2H),2.26-2.14 (m, 1H), 1.92-1.72 (m, 3H), 1.39 (s, 9H), 1.05-0.95 (m, 1H)).

EXAMPLE 36 (2S,3S)-3-(Acetylamino-methyl)-pyrrolidine-1,2-dicarboxylicacid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

Triethylamine (0.339 mmol) was added to a solution ofimidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A) (0.113 mmol) and(2S,3S)-3-(acetylamino-methyl)-pyrrolidine-2-carboxylic acid amide(Stage 36.1) (0.135 mmol) at rt. After stirring for 85 min, the reactionmixture was concentrated. The residue was purified by silica gel columnchromatography followed by trituration with Et₂O to afford the titlecompound as a yellow solid. HPLC: t_(R)=4.20 min (method H); LCMS:t_(R)=1.48 min, [M+H]⁺ 472 (method I); TLC: R_(f)=0.14 (9:1CH₂Cl₂/MeOH); ¹H-NMR (d₆-DMSO, 600 MHz): 11.15 (br s, 1H), 8.41 (s, 1H),7.81 (br s, 1H), 7.53 (br s, 1H), 7.13 (br s, 1H), 4.28 (m, 1H), 3.74(m, 1H), 3.40 (m, 1H), 3.25 (m, 1H), 2.98 (m, 2H), 2.93 (m, 3H), 2.42(m, 1H), 2.02 (m, 1H), 1.82 (s, 3H), 1.74 (m, 1H), 1.33 (s, 9H).

Stage 36.1: (2S,3S)-3-(Acetylamino-methyl)-pyrrolidine-2-carboxylic acidamide

A mixture of(2S,3S)-3-(acetylamino-methyl)-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide (Stage 36.2) (0.48 mmol) and 10% Pd on charcoal, wet with 50%H₂O (Aldrich 330108) (0.096 mmol) in MeOH (5 mL) was hydrogenated for6.5 h at rt. The reaction mixture was then filtered through a FluoroporeMembrane Filter (0.2 μm FG) and evaporated. The residue was dissolved inCH₂Cl₂ and evaporated to dryness to afford the title compound as anoff-white solid. ESI-MS: [M+H]⁺ 186; TLC: R_(f)=0.08 (200:20:1CH₂Cl₂/MeOH/conc. NH₄OH).

Stage 36.2:(2S,3S)-3-(Acetylamino-methyl)-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide

Thioacetic acid (2.312 mmol) was added to(2S,3S)-3-azidomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide (Stage 36.3) (0.578 mmol) at rt with the formation ofnitrogen gas. After stirring for 16 h, the reaction mixture was dilutedwith Et₂O, the solids were removed by filtration and the filtrate wasconcentrated. The residue was purified by silica gel columnchromatography to afford the title compound as a light yellow oil (thiolodor). HPLC: t_(R)=3.71 min (method H); LC-MS: t_(R)=0.64 min, [M+H]⁺290 (method I); TLC: R_(f)=0.38 (200:20:1 CH₂Cl₂/MeOH/conc. NH₄OH).

Stage 36.3:(2S,3S)-3-Azidomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide

Trimethylaluminum in toluene (2 M, 15.95 mmol) was added dropwise to amixture of NH₄Cl (15.95 mmol) in toluene (2 mL) at 0° C. with theformation of methane gas. The reaction mixture was allowed to warm tort, stirred for a further 15 min and then slowly treated with a solutionof (2S,3S)-3-azidomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester (Stage 36.4) (7.98 mmol) in toluene (8 mL). Additionalreagent prepared from NH₄Cl (15.95 mmol) in toluene (2 mL) andtrimethylaluminum in toluene (2 M, 15.95 mmol) at 0° C. was added after18 h. After stirring for 44 h, the mixture was cooled to 0° C., quenchedwith 1M HCl and then washed with CH₂Cl₂ (3×). The aqueous phase wasbasified with a 1:1 saturated solution of NaHCO₃/saturated solution ofRochelle's salt and extracted with THF (10×). The combined organiclayers were dried (Na₂SO₄), filtered and concentrated. The residue waspurified using a RediSep® silica gel column to afford the title compoundas a yellow oil. HPLC: t_(R)=2.50 min (method G); ESI-MS: [M+H]⁺ 274;TLC: R_(f)=0.26 (3:1 Hex/EtOAc).

Stage 36.4:(2S,3S)-3-Azidomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester

Sodium azide (5.34 mmol) was added to a solution of(2S,3R)-3-iodomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester (Stage 36.5) (3.56 mmol) in DMF (30 mL) at rt. After18 h, the reaction mixture was poured onto water and extracted with MTBE(2×). The combined organic phases were washed with brine, dried(Na₂SO₄), filtered and concentrated. The residue was purified using aRediSep° silica gel column to to afford the title compound as a brownoil. HPLC: t_(R)=3.26 min (method G); ESI-MS: [M+H]⁺ 289.

Stage 36.5:(2S,3R)-3-Iodomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester

A solution of [but-3-enyl-((S)-1-phenyl-ethyl)-amino]-acetic acid methylester [432555-77-6] (20.22 mmol) in THF (10 mL) was slowly added to asolution of lithium diisopropylamide (24.26 mmol) in 1:2 hexanes/THF (30mL) at −78° C. The reaction mixture was warmed to 0° C., stirred for 1 hand then re-cooled to −78° C. A solution of zinc bromide (50.5 mmol) inEt₂O (40 mL) was added and the reaction mixture was then warmed to rt.After stirring for 1 h, the mixture was cooled to 0° C. and iodine(22.24 mmol) was added in portions. The reaction mixture was stirred at0° C. for 2 h and at rt for another 2 h, diluted with Et₂O and thensuccessively washed with a saturated solution of Na₂S₂O₃ and a saturatedsolution of NH₄Cl. The aqueous layers were each back-extracted withEt₂O. The combined organic phases were dried (Na₂SO₄), filtered andconcentrated. The residue was purified by silica gel columnchromatography to afford the title compound as a red oil. HPLC:t_(R)=3.46 min (method G); ESI-MS: [M+H]⁺ 374.

EXAMPLE 37 (2S,3S)-3-Morpholin-4-ylmethyl-pyrrolidine-1,2-dicarboxylicacid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

Triethylamine (0.423 mmol) was added to a solution ofimidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A) (0.141 mmol) and(2S,3S)-3-(acetylamino-methyl)-pyrrolidine-2-carboxylic acid amide(Stage 37.1) (0.155 mmol) in DMF (0.5 mL) at rt. After stirring for 3 h,the reaction mixture was concentrated. The residue was purified bysilica gel column chromatography to afford the title compound as a whitesolid. HPLC: t_(R)=4.04 min (method H); LCMS: t_(R)=1.36 min, [M+H]⁺ 500(method I); TLC: R_(f)=0.09 (19:1 CH₂Cl₂/MeOH); ¹H-NMR (d₆-DMSO, 600MHz): 11.15 (br s, 1H), 8.39 (m, 1H), 7.37 (br s, 1H), 7.05 (s, 1H),4.27 (m, 1H), 3.70 (m, 1H), 3.59 (m, 4H), 3.42 (m, 1H), 2.97 (m, 2H),2.90 (m, 2H), 2.58 (m, 1H), 2.38 (m, 4H), 2.35 (m, 1H), 2.17 (m, 1H),2.02 (m, 1H), 1.75 (m, 1H), 1.32 (s, 9H).

Stage 37.1: (2S,3S)-3-Morpholin-4-ylmethyl-pyrrolidine-2-carboxylic acidamide

A mixture of(2S,3S)-3-morpholin-4-ylmethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide (Stage 37.2) (1.046 mmol) and 10% Pd on charcoal, wet with50% H₂O (Aldrich 330108) (0.105 mmol) in MeOH (5 mL) was hydrogenatedfor 6.5 h at rt. The reaction mixture was then filtered through aFluoropore Membrane Filter (0.2 μm FG) and evaporated. The residue wasdissolved in CH₂Cl₂ and evaporated to dryness to afford the titlecompound as colorless oil. ESI-MS: [M+H]⁺ 214; TLC: R_(f)=0.14 (200:20:1CH₂Cl₂/MeOH/conc. NH₄OH).

Stage 37.2:(2S,3S)-3-Morpholin-4-ylmethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide

Trimethylaluminum in toluene (2 M, 2.89 mmol) was added dropwise to amixture of NH₄Cl (2.89 mmol) in toluene (3 mL) at 0° C. with theformation of methane gas. The reaction mixture was allowed to warm tort, stirred for a further 15 min and then slowly treated with a solutionof(2S,3S)-3-morpholin-4-ylmethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester (Stage 37.3) (1.444 mmol) in toluene (9 mL).Additional reagent prepared from NH₄Cl (2.89 mmol) in toluene (2 mL) andtrimethylaluminum in toluene (2 M, 2.89 mmol) at 0° C. was added after18 h. After stirring for 60 h, the mixture was cooled to 0° C., quenchedwith 1M HCl and then washed with CH₂Cl₂ (3×). The aqueous phase wasbasified with a 1:1 saturated solution of NaHCO₃/saturated solution ofRochelle's salt and extracted with THF (10×). The combined organiclayers were dried (Na₂SO₄), filtered and concentrated. The residue waspurified by silica gel column chromatography to afford the titlecompound as a yellow oil. HPLC: t_(R)=2.18 min (method G); ESI-MS:[M+H]⁺ 318.

Stage 37.3:(2S,3S)-3-Morpholin-4-ylmethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester

A mixture of(2S,3R)-3-iodomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester (Stage 36.5) (7.07 mmol), K₂CO₃ (21.22 mmol) andmorpholine (10.61 mmol) in CH₃CN (24 mL) was stirred at 50° C. for 62 h.The reaction mixture was poured onto ice water and extracted with EtOAc(3×). The combined organic layers were successively washed with waterand brine, dried (Na₂SO₄), filtered and concentrated. The residue waspurified using a RediSep® silica gel column to afford the title compoundas a yellow oil. HPLC: t_(R)=2.56 min (method G); ESI-MS: [M+H]⁺ 333.

EXAMPLE 38 (2S,3R)-3-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

The title compound was synthesized using methodology as described forExample 20 using (2S,3R)-3-hydroxy-pyrrolidine-2-carboxylic acid amide(H. Fukushima et al. Bioorg. Med. Chem. 2004, 12, 6053; H. Ji et al. J.Med. Chem. 2006, 49(21), 6254) instead of intermediate P.

LCMS: t_(R)=1.45 min, M+H=417.0, M−H=415 (method J). ¹H-NMR (d₆-DMSO,600.13 MHz): 12-11 (s, br, 1H) 8.4 (s, 1H), 7.2-6.9 (m, 2H), 5.2 (s,1H), 4.4 (s, 1H), 4.25 (s, br, 1H), 3.6 (m, 1H); 3.45 (m, 1H), 2.95 (m,2H), 2.9 (m, 2H), 2.0 (m, 1H), 1.9 (m, 1H), 1.3 (s, 9H).

EXAMPLE 39 (2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

The title compound was synthesized using methodology as described forExample 34 using (2S,3R)-3-methyl-pyrrolidine-2-carboxylic acid amide(intermediate P) instead of intermediate D.

LCMS: t_(R)=1.32 min, M+H=420.0, M−H=418.1 (method J). ¹H-NMR (d₆-DMSO,400 MHz): 10.65 (s, br, 1H) 7.39 (s, br, 1H), 6.99 (s, br, 1H), 4.2 (m,1H), 3.67 (t, 1H), 3.3 (m, 1H), 3.08 (t, 2H), 2.93 (t, 2H), 2.38 (m,1H), 1.95 (m, 1H), 1.7 (m, 1H), 1.39 (s, 9H), 0.97 (d 3H).

Synthesis Scheme:

EXAMPLE 40 (2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-methyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-amide]

The title compound was prepared starting from imidazole-1-carboxylicacid(8-methyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-amide(Stage 40.1) using synthetic methodology described in the preparation ofExample 20.

LCMS: t_(R)=0.29 min, M+H=374.0, M−H=372 (method J). ¹H-NMR 1H-NMR(CD₃OD, 400 MHz): 7.9 (s, 1H), 7.0 (s, 1H), 5.27 (s, 2H), 4.38 (d, 1H),3.8 (dd, 1H), 3.5 (m, 1H), 2.5 (m, 1H), 2.4 (s, 1H), 2.08 (m, 1H), 1.95(m, 1H), 1.12 (d, 3H).

Stage 40.1: Imidazole-1-carboxylic acid(8-methyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-amide

Imidazole-1-carboxylic acid(8-methyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-amidewas prepared fromN-(8-methyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-acetamide(Stage 40.2) using synthetic methodology as described for thepreparation of intermediate A.

Stage 40.2:N-(8-methyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-acetamide

A mixture of 463 mg (1.53 mmol)N-[4-(4-bromo-6-methyl-pyridin-3-yloxymethyl)-thiazol-2-yl]-acetamide(Stage 40.3), 900 mg (2.71 mmol) cesium carbonate, 30.4 mg (0.135 mmol)palladium acetate and 81 mg (0.271 mmol) tributylphosphinetetrafluoroborate in 3 mL DMF was stirred at 115° C. for 7 h underargon. Then the reaction mixture was poured onto water, EtOAc was addedand, after filtration over a layer of Hyflo Super Gel medium (Fluka56678), the filtrate was extracted with EtOAc (2×). The combined organiclayers were washed with water and brine, dried (MgSO₄) and filtered. Thefiltrate was concentrated in vacuo to leave a residue that was purifiedon silica gel (=−100% EtOAc in heptane) to afford 93 mg of the titlecompound as a solid. LCMS: t_(R)=0.27 min, M+H=262, M−H 260 (method J).

Stage 40.3:N-[4-(4-Bromo-6-methyl-pyridin-3-yloxymethyl)-thiazol-2-yl]-acetamide

To a solution of 270 mg (1.44 mmol) 4-bromo-6-methyl-pyridin-3-ol (Stage40.4) in DMF (5 mL), 44.8 mg NaH (1.87 mmol) was added and this mixturewas stirred for 1 h at rt. 301 mg (1.58 mmol)2-acetamido-4-(chloromethyl)-1,3-thiazole (Apollo OR15549) was added andstirring was continued at rt for another 17 h. The reaction mixture waspoured on water and extracted with EtOAc (2×). The combined organiclayers were washed with water and brine, dried (MgSO₄) and filtered. Thefiltrate was concentrated in vacuo to leave 350 mg of the title compound(white solid) considered sufficiently pure without further purification.LCMS: t_(R)=1.36 min, M+H=342 (⁷⁹Br), 344 (⁸¹Br) (method J).

Stage 40.4: 4-Bromo-6-methyl-pyridin-3-ol

920 mg (3.05 mmol) 4-bromo-5-methoxymethoxy-2-methyl-pyridine (Stage40.5) were dissolved in 5 mL MeOH, HCl conc. (1 mL, 12 mmol) was addedand the reaction mixture was stirred for 2 h at rt. While concentratingthe mixture in vacuo, the title compound (as hydrochloride)crystallized. Filtration afforded 570 mg white crystals. ¹H-NMR(d₆-DMSO, 400 MHz): 8.19 (s, 1H), 8.04 (s, 1H), 2.53 (s, 3H).

Stage 40.5: 4-Bromo-5-methoxymethoxy-2-methyl-pyridine

A solution of 1000 mg (6.53 mmol) 5-methoxymethoxy-2-methyl-pyridine(J.-P. Behr et al. Bioorg. Med. Chem. Lett. 2003, 13(10), 1713) in 10 mLTHF was cooled to −78° C., where 4.03 mL (6.85 mmol) t-BuLi (1.7Msolution in pentane) was added. The resulting mixture was stirred underargon for 1 h, then 2.126 g (6.53 mmol) 1,2-dibromotetrachloroethane (in5 mL THF) was added. Stirring was continued for 1 h at −78° C. and thereaction mixture was warmed to rt. Saturated NH₄Cl solution was addedand the aqueous mixture was extracted with EtOAc (2×). The combinedorganic layers were washed with H₂O and brine, dried (MgSO₄) andfiltered. The filtrate was concentrated in vacuo to leave a residue thatwas purified on silica gel (EtOAc/hexane=1:1) to afford 920 mg of thetitle compound as an oil. LCMS: t_(R)=0.29 min, M+H=232 (⁷⁹Br), 234(⁸¹Br) (method J).

EXAMPLE 41 (2S,3S)-3-Morpholin-4-ylmethyl-pyrrolidine-1,2-dicarboxylicacid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

The title compound was synthesized using methodology as described forExample 34 using (2S,3S)-3-morpholin-4-ylmethyl-pyrrolidine-2-carboxylicacid amide (Stage 37.1) instead of intermediate D.

LCMS: t_(R)=0.68 min, M+H=505.3.0, M−H=503.2 (method J). ¹H-NMR(d₆-DMSO, 600.13 MHz): 10.65 (s, b, 1H), 7.4 (s, b, 1H), 7.05 (s, b,1H), 4.3 (s, b, 1H), 3.7 (m, 1H), 3.6 (m, 4H), 3.3 (m, 1H), 3.1 (t, 2H),2.9 (t, 2H), 2.6 (m, 1H), 2.4 (m, 1H), 2.35 (m, 4H), 2.15 (m, 1H), 2.0(m, 1H), 1.75 (m, 1H), 1.35 (s, 9H).

EXAMPLE 42 (2S,3R)-3-Methoxymethyl-pyrrolidine-1,2-dicarboxylic acid2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

Triethylamine (0.525 mmol) was added to a solution ofimidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A) (0.175 mmol) and(2S,3R)-3-methoxymethyl-pyrrolidine-2-carboxylic acid amide (Stage 42.1)(0.192 mmol) in DMF (0.7 mL) at rt. After stirring for 3 h, the reactionmixture was concentrated. The residue was purified by silica gel columnchromatography to afford the title compound as a white solid. HPLC:t_(R)=2.84 min (method G); LCMS: t_(R)=1.71 min, [M+H]⁺ 445 (method I);TLC: R_(f)=0.41 (19:1 CH₂Cl₂/MeOH); ¹H-NMR (d₆-DMSO, 600 MHz): 11.15 (brs, 1H), 8.40 (s, 1H), 7.42 (br s, 1H), 7.05 (br s, 1H), 4.27 (m, 1H),3.74 (m, 1H), 3.46 (m, 1H), 3.42 (m, 1H), 3.24 (s, 3H), 3.16 (m, 1H),2.98 (m, 2H), 2.91 (m, 2H), 2.54 (m, 1H), 2.03 (m, 1H), 1.77 (m, 1H),1.32 (s, 9H).

Stage 42.1: (2S,3R)-3-Methoxymethyl-pyrrolidine-2-carboxylic acid amide

The title compound was prepared in analogy to the procedure described inStage 37.1 but(2S,3R)-3-methoxymethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide (Stage 42.2) was used instead of(2S,3S)-3-morpholin-4-ylmethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide.

The title compound was obtained as a white solid. ESI-MS: [M+H]⁺ 159.

Stage 42.2:(2S,3R)-3-Methoxymethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide

The title compound was prepared in analogy to the procedure described inStage 37.2 but(2S,3R)-3-methoxymethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester (Stage 42.3) was used instead of(2S,3S)-3-morpholin-4-ylmethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester.

The title compound was obtained as a yellow oil. HPLC: t_(R)=2.35 min(method G); LC-MS: t_(R)=0.47 min, [M+H]⁺ 263 (method K); TLC:R_(f)=0.05 (1:1 Heptanes/EtOAc).

Stage 42.3:(2S,3R)-3-Methoxymethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid methyl ester

A mixture of(3aR,6aS)-1-((S)-1-phenyl-ethyl)-hexahydro-furo[3,4-b]pyrrol-6-one[805246-48-4] (17.05 mmol), KOH (71.60 mmol) and iodomethane (68.20mmol) in toluene (79 mL) was stirred at 80° C. for 1.5 h. The reactionmixture was cooled to rt and partitioned between water and MTBE. Theaqueous layer was extracted with MTBE (3×). The combined organic layerswere dried (Na₂SO₄), filtered and concentrated. The residue was purifiedby silica gel column chromatography to afford the title compound as ayellow oil. HPLC: t_(R)=2.98 min (method G); LC-MS: t_(R)=0.69 min,[M+H]⁺ 278 (method K); TLC: R_(f)=0.25 (1:3 Heptanes/EtOAc).

EXAMPLE 43 (2S,3S)-3-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylicacid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide]

Triethylamine (0.305 mmol) was added to a solution ofimidazole-1-carboxylic acid(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide(Intermediate A) (0.102 mmol) and(2S,3S)-3-dimethylaminomethyl-pyrrolidine-2-carboxylic acid amide (Stage43.1) (0.102 mmol) in DMF (0.3 mL) at rt. After stirring for 0.5 h, thereaction mixture was concentrated and dried overnight under vacuum at50° C. The residue was suspended in EtOAc (1 mL), filtered and driedunder vacuum to afford the title compound as a white solid. HPLC:t_(R)=4.01 min (method H); LCMS: t_(R)=1.33 min, [M+H]⁺ 458 (method I);TLC: R_(f)=0.08 (4:1 CH₂Cl₂/MeOH); ¹H-NMR (d₆-DMSO, 600 MHz): 11.14 (brs, 1H), 8.40 (s, 1H), 7.42 (br s, 1H), 7.06 (br s, 1H), 4.27 (m, 1H),3.71 (m, 1H), 3.42 (m, 1H), 2.98 (m, 2H), 2.90 (m, 2H), 2.52 (m, 1H),2.33 (m, 1H), 2.18 (m, 1H), 2.18 (s, 6H), 2.01 (m, 1H), 1.72 (m, 1H),1.32 (s, 9H).

Stage 43.1: (2S,3S)-3-Dimethylaminomethyl-pyrrolidine-2-carboxylic acidamide

The title compound was prepared in analogy to the procedure described inStage 37.1 but(2S,3S)-3-dimethylaminomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide (Stage 43.2) was used instead of(2S,3S)-3-morpholin-4-ylmethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide. Also, the hydrogenation was performed under 4 bar pressure.

The title compound was obtained as a yellow oil. ESI-MS: [M+H]⁺ 172.

Stage 43.2:(2S,3S)-3-Dimethylaminomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide

A mixture of(2S,3S)-3-aminomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide (Stage 43.3) (0.418 mmol), sodium cyanoborohydride (2.86mmol) and 37% aqueous formaldehyde (2.14 mmol) in MeOH (3.3 mL) wasstirred at 55° C. for 16 h. The reaction mixture was cooled to rt andconcentrated. The residue was purified using a RediSep® silica gelcolumn to afford the title compound as a white foam. HPLC: t_(R) 3.59min (method H); LC-MS: t_(R)=0.86 min, [M+H]⁺ 276 (method I); TLC:R_(f)=0.13 (9:1 CH₂Cl₂/MeOH).

Stage 43.3:(2S,3S)-3-Aminomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide

A mixture of(2S,3S)-3-azidomethyl-1-((S)-1-phenyl-ethyl)-pyrrolidine-2-carboxylicacid amide (Stage 36.3) (0.723 mmol) and triphenylphosphine (0.867 mmol)in THF (3 mL) was stirred at rt for 25 h. The reaction mixture wasconcentrated to afford the crude title compound as a light brown solid.HPLC: t_(R) 3.53 min (method H); ESI-MS: [M+H]⁺ 248.

EXAMPLE 44 (2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[8-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl]-amide}

Triethylamine (1.714 mmol) was added to a solution ofimidazole-1-carboxylic acid[8-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl]-amide(Stage 44.1) (0.490 mmol) and (2S,3R)-3-methyl-pyrrolidine-2-carboxylicacid amide (intermediate P) (0.979 mmol) in DMF (1.5 mL) at rt. Afterstirring for 1.5 h, the reaction mixture was concentrated. The residuewas diluted with a saturated solution of NaHCO₃ and extracted with EtOAc(2×). The combined organic layers were successively washed with waterand brine, dried (Na₂SO₄), filtered and concentrated. The residue waspurified using a RediSep® silica gel column to to afford the titlecompound as a beige solid. HPLC: t_(R)=5.77 min (method H); LCMS:t_(R)=2.18 min, [M+H]⁺ 469 (method I); TLC: R_(f)=0.16 (19:1CH₂Cl₂/MeOH); ¹H-NMR (d₆-DMSO, 600 MHz): 11.19 (br s, 1H), 8.50 (s, 1H),7.43 (br s, 1H), 7.01 (br s, 1H), 4.18 (m, 1H), 3.72 (m, 1H), 3.40 (m,1H), 3.03 (m, 2H), 2.94 (m, 2H), 2.38 (m, 1H), 1.97 (m, 1H), 1.70 (m,1H), 1.58 (s, 6H), 0.99 (d, 3H).

Stage 44.1: Imidazole-1-carboxylic acid[8-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl]-amide

The title compound was prepared in analogy to the procedure describedfor Intermediate A but in Stage A.33,3,3-trifluoro-2,2-dimethyl-propionamidine hydrochloride (Stage 44.2)was used instead of tert-butylamidine hydrochloride.

The title compound was obtained as a beige solid. HPLC: t_(R)=6.73 min(method H); LCMS: t_(R)=1.11 min, [M+H]⁺ 373 (method J). Note: forcharacterization purposes, the title compound was dissolved inMeOH→methyl carbamate derivative.

Stage 44.2: 3,3,3-Trifluoro-2,2-dimethyl-propionamidine hydrochloride

3,3,3-Trifluoro-2,2-dimethyl-propionitrile (Stage 44.3) (12.40 mmol) wasadded to a solution of sodium methoxide (freshly prepared from 48.40mmol sodium metal and 102 mL MeOH) at rt. After 4 h, acetic acid (48.40mmol) and then NH₄Cl (14.88 mmol) were slowly added and the reactionmixture was then heated to 70° C. for 40 h. After concentrating (40° C.,100 mbar) the reaction mixture, the residue was suspended in acetone,filtered and dried under vacuum to afford the title compound as a whitesolid. ESI-MS: [M+H]⁺ 155.

Stage 44.3: 3,3,3-Trifluoro-2,2-dimethyl-propionitrile

A mixture of 3,3,3-trifluoro-2,2-dimethyl-propionamide (Stage 44.4)(121.2 mmol) and phosphorus pentoxide (121.2 mmol) was slowly heated to200° C. and the resulting distillate collected. The title compound wasobtained as a colorless liquid.

Stage 44.4: 3,3,3-Trifluoro-2,2-dimethyl-propionamide

Oxalyl chloride (140.8 mmol) was added dropwise to a solution of3,3,3-trifluoro-2,2-dimethyl-propionic acid [889940-13-0] (128 mmol) inCH₂Cl₂ (128 mL) at 0° C. Added a few dr DMF until gas evolution wasobserved and then continued stirring for 30 min. After warming to rt andstirring overnight, the reaction mixture was concentrated (40° C., 00mbar). The residue was dissolved in THF (128 mL), cooled to 0° C. andthen slowly treated with a solution of concentrated aqueous ammonia (64mL). After stirring at 0° C. for 30 min and then at rt for 4 h, thereaction mixture was concentrated to half its volume to give a thickwhite suspension. After filtering and drying, the title compound wasobtained as a white solid. ESI-MS: [M+H]⁺ 156.

EXAMPLE 45 (2S,3R)-3-Methoxymethyl-pyrrolidine-1,2-dicarboxylic acid2-amide1-{[8-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl]-amide}

Triethylamine (1.714 mmol) was added to a solution ofimidazole-1-carboxylic acid[8-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl]-amide(Stage 44.1) (0.490 mmol) and(2S,3R)-3-methoxymethyl-pyrrolidine-2-carboxylic acid amide (Stage 42.1)(0.0.979 mmol) in DMF (1.5 mL) at rt. After stirring for 2 h, thereaction mixture was concentrated. The residue was diluted with asaturated solution of NaHCO₃ and extracted with EtOAc (2×). The combinedorganic layers were successively washed with water and brine, dried(Na₂SO₄), filtered and concentrated. The residue was purified using aRediSep® silica gel column to to afford the title compound as a beigesolid. HPLC: t_(R)=5.71 min (method H); LCMS: t_(R)=2.15 min, [M+H]⁺ 499(method I); TLC: R_(f)=0.40 (19:1 CH₂Cl₂/MeOH); ¹H-NMR (d₆-DMSO, 600MHz): 11.23 (br s, 1H), 8.50 (s, 1H), 7.42 (br s, 1H), 7.06 (br s, 1H),4.27 (m, 1H), 3.74 (m, 1H), 3.46 (m, 1H), 3.42 (m, 1H), 3.24 (s, 3H),3.16 (m, 1H), 3.03 (m, 2H), 2.94 (m, 2H), 2.55 (m, 1H), 2.03 (m, 1H),1.77 (m, 1H), 1.58 (s, 6H).

EXAMPLE 46 (2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-amide]

LCMS: t_(R)=0.50 min, M+H=416, M−H=414 (method L). ¹H-NMR (d₆-DMSO, 400MHz): 8.05 (s, 1H), 7.37 (bs, 1H), 7.09 (s, 1H), 6.97 (bs, 1H), 5.31 (s,2H), 4.18 (m, 1H), 3.69 (dd, 1H), 3.39 (m, 1H), 2.35 (m, 1H), 1.95 (m,1H), 1.71 (m, 1H), 1.27 (s, 9H), 0.975 (d, 3H).

The title compound was prepared from2-tert-butyl-5-methoxymethoxy-pyridine using synthetic methodology asdescribed for the preparation of Example 40. The starting material,2-tert-butyl-5-methoxymethoxy-pyridine was prepared from2-bromo-5-methoxymethoxy-pyridine as described below:

2-tert-Butyl-5-methoxymethoxy-pyridine

6.69 g (74.7 mmol) copper(I)cyanide in 15 ml dry THF was cooled to −75°C., where 149 ml (149 mmol) of a 1M tert-butylmagnesium chloridesolution was added drop-wise. Stirring was continued for 40 min. Afterthat, 4.07 g (18.67 mmol) 2-bromo-5-methoxymethoxy-pyridine (Zhong, W.et al. WO2008147547) dissolved in 30 ml dry THF were added drop-wise.The reaction mixture was stirred at −75° C. for 1 h and then allowed toreach room temperature, where stirring was continued for another 6 h.Then 30 ml of 25% aqueous ammonia was added to the reaction mixture. Themixture was filtered and extracted twice with CH₂Cl₂. The combinedorganic layers were washed with water, dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by chromatography onsilicagel (EtOAc, heptane) to yield 2.64 g of the pure title compound asan oil. LCMS: t_(R)=0.55 min, M+H=196 (method K).

EXAMPLE 47 (2S,3S)-3-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylicacid 2-amide1-[(8-tert-butyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-amide]

The title compound was prepared using synthetic methodology describedfor the preparation of Example 46 and using(2S,3S)-3-dimethylaminomethyl-pyrrolidine-2-carboxylic acid amide (Stage43.1) instead of (2S,3R)-3-methyl-pyrrolidine-2-carboxylic acid amide inthe last step of the synthesis.

LC-MS: t_(R)=0.24 min, M−H=457 (method L). ¹H-NMR (d₆-DMSO, 400 MHz):11.15 (bs, 1H), 8.03 (s, 1H), 7.35 (s, 1H), 7.07 (s, 1H), 7.0 (s, 1H),5.28 (s, 2H), 4.23 (m, 1H), 3.65 (m, 1H), 3.40 (m, 1H), 2.6-2.45 (m,2H), 2.30 (m, 1H), 2.17 (s, 6H), 2.15 (m, 1H), 1.70 (m, 1H), 1.27 (s,9H).

EXAMPLE 48 (2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[8-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl]-amide}

LC-MS: t_(R)=1.59 min, M+H=470 (method F). ¹H-NMR (d₆-DMSO, 400 MHz):11.25 (bs, 1H), 8.15 (s, 1H), 7.42 (s, 1H), 7.34 (s, 1H), 7.00 (s, 1H),5.40 (s, 2H), 4.19 (s, 1H), 3.71 (m, 1H), ca. 3.4 (m, 1H), 2.5-2.35 (m,1H), 1.99 (m, 1H), 1.68 (m, 1H), 1.56 (s, 6H), 0.99 (d, 3H).

The title compound was prepared starting from4-bromo-5-methoxymethoxy-2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridineusing synthetic methodology as described for the preparation of Examples40 and 42.

4-Bromo-5-methoxymethoxy-2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridinewas Prepared as Follows

a)4-Bromo-5-methoxymethoxy-2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridine

The title compound was prepared using synthetic methodology as describedfor the preparation of Example 40, stage 40.5. as an oil. LCMS (methodL): t_(R)=1.26 min, M+H=328 (⁷⁹Br), 330 (⁸¹Br).

b) 5-Methoxymethoxy-2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridine

1.7 g (4.13 mmol) methanesulfonic acid2,2,2-trifluoro-1-(5-methoxymethoxy-pyridin-2-yl)-1-methyl-ethyl esterwas dissolved in 20 ml dichloromethane. The reaction mixture was cooledto 0° C. where 2.065 ml (4.13 mmol) trimethylaluminum was added slowly.The reaction mixture was stirred at room temperature for 20 h. Afterthat water was added slowly and the mixture was extracted 2 times withEtOAc. The combined organic layers were washed with water and brine,dried (MgSO₄), filtered and concentrated in vacuo. The residue waspurified by chromatography on silicagel (EtOAc, heptane) to yield toafford 0.55 g of the pure title compound. LCMS (method L): t_(R)=1.09min, M+H=250.

c) Methanesulfonic acid2,2,2-trifluoro-1-(5-methoxymethoxy-pyridin-2-yl)-1-methyl-ethyl ester

To a stirred mixture of 0.213 g (8.44 mmol) sodium hydride in 6 ml dryTHF, 1.06 g (4.22 mmol)1,1,1-trifluoro-2-(5-methoxymethoxy-pyridin-2-yl)-propan-2-ol (dissolvedin 4.9 ml dry THF) was added dropwise at 0° C. After completion of theaddition, stirring was continued for 3 h at room temperature. Thenmethanesulfonyl chloride (dissolved in 7.3 ml dry THF) was added at roomtemperature and stirring was continued for another 2 h. Then water andsat. NaHCO₃ soln. were added. This mixture was extracted 2 times withEtOAc. The combinded organic layers were washed with water and brine,dried (MgSO₄), filtered and concentrated in vacuo to afford 1.7 g of thetitle compound, which was used directly in the next step. LCMS (methodL): t_(R)=0.96 min, M+H=330.

d) 1,1,1-Trifluoro-2-(5-methoxymethoxy-pyridin-2-yl)-propan-2-ol

A mixture of 1.48 g (4.58 mmol)5-methoxymethoxy-2-(2,2,2-trifluoro-1-methyl-1-trimethylsilanyloxy-ethyl)-pyridineand 5 ml (10 mmol) 2N HCl in 15 ml THF was stirred at room temperaturefor 2 h. After that the reaction mixture was poured on water and a pH of1-2 was adjusted by the addition of 2N HCl. The reaction mixture wasextracted 2 times with EtOAc. The combinded organic layers were washedwith water and brine, dried (MgSO₄), filtered and concentrated in vacuoto afford 1.06 g of the title compound as an oil (88% pure), which wasused in the next step without purification. LCMS (method L): t_(R)=0.88min, M+H=252.

e)5-Methoxymethoxy-2-(2,2,2-trifluoro-1-methyl-1-trimethylsilanyloxy-ethyl)-pyridine

A mixture of 1.12 g (6.18 mmol)1-(5-methoxymethoxy-pyridin-2-yl)-ethanone, 1.055 g (7.42 mmol)trimethyl(trifluormethyl)silane and 0.025 g (0.309 mmol) sodium acetatein 9 ml DMF was stirred for 1 h at 0° C. and 1 h at room temperature.After that the reaction mixture was poured on water and a pH of 1-2 wasadjusted by the addition of 2N HCl. The reaction mixture was extracted 2times with EtOAc. The combinded organic layers were washed with waterand brine, dried (MgSO₄), filtered and concentrated in vacuo. Theresidue was purified by chromatography on silicagel (EtOAc, heptane) toyield 1.87 g of the pure title compound as an oil. LCMS (method L):t_(R)=1.38 min, M+H=324.

f) 1-(5-Methoxymethoxy-pyridin-2-yl)-ethanone

NaH was added to a solution of 1 g (7.29 mmol)1-(5-hydroxy-pyridin-2-yl)-ethanone (Anichem) in 15 ml DMF at 0° C.After stirring this mixture for 1 h, 0.78 ml (8.75 mmol) MOMCl was addedat 0° C. Then the reaction mixture was stirred at room temperature for 2h. The reaction mixture was poured on water and extracted 2 times withEtOAc. The combinded organic layers were washed with water and brine,dried (MgSO₄), filtered and concentrated in vacuo. The residue waspurified by chromatography on silicagel (EtOAc, heptane) to yield 1.12 gof the pure title compound as an oil. LCMS (method L): t_(R)=0.66 min,M+H=182.

EXAMPLE 49 (2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[7-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl]-amide}

LCMS: t_(R)=0.97 min, M+H=474, M−H=414 (method L). ¹H-NMR (d₆-DMSO, 400MHz): 10.7 (bs, 1H), 7.37 (s, 1H), 6.97 (s, 1H), 4.18 (m, 1H), 3.69 (dd,1H), 3.39 (m, 1H), 3.14 (t, 2H), 2.95 (t, 2H), 2.34 (m, 1H), 1.94 (m,1H), 1.68 (m, 1H), 1.62 (s, 6H), 0.965 (s, 3H). The title compound wasprepared from 2,2-dimethyl-thiopropionamide using synthetic methodologyas described for the preparation of Example 39. The starting material,3,3,3-trifluoro-2,2-dimethyl-thiopropionamide was prepared as describedbelow:

Preparation of 3,3,3-trifluoro-2,2-dimethyl-thiopropionamide a)3,3,3-Trifluoro-2,2-dimethyl-propionamide

A mixture of 2 g (12.81 mmol) 3,3,3-trifluoro-2,2-dimethylpropionic acid(Aldrich), 2.077 g (12.81 mmol) carbonyl-diimidazole and 5.55 ml (64.1mmol) aqueous ammonia in 50 ml CH₂Cl₂ was stirred at room temperaturefor 20 h. After that, diethyl ether was added and the resulting mixturewas washed with 0.1 N HCL, 0.1 N NaOH, water and brine. The organiclayer was dried (MgSO₄), filtered and concentrated in vacuo to afford1.15 g of the title compound as white crystals, that were used in thenext step without further purification. MS: M+H=156.

b) 3,3,3-Trifluoro-2,2-dimethyl-thiopropionamide

A mixture of 1.05 g (6.77 mmol)3,3,3-trifluoro-2,2-dimethyl-propionamide and 1.48 g (3.66 mmol)Lawesson's reagent in 32 ml dry THF was stirred at reflux temperaturefor 20 h. After that the reaction mixture was cooled to room temperatureand concentrated in vacuo. The residue was purified by chromatography onsilicagel (heptane, EtOAc) to afford 0.81 g of the title compound aspure white crystals. MS: M+H=172.

Using (2S,3R)-3-methoxymethyl-pyrrolidine-2-carboxylic acid amide (Stage42.1) in the last step of the synthesis the following example wasprepared in the same way:

EXAMPLE 50 (2S,3R)-3-Methoxymethyl-pyrrolidine-1,2-dicarboxylic acid2-amide1-{[7-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl]-amide}

LC-MS: t_(R)=0.96 min, M+H=504, M−H=502 (method L). ¹H-NMR (d₆-DMSO, 400MHz): 10.76 (bs, 1H), 7.34 (bs, 1H), 7.02 (bs, 1H), 4.29 (m, 1H), 3.45(m, 1H), 3.42 (m, 2H), 3.22 (s, 3H), 3.14 (m, 3H), 2.97 (m, 2H), 2.48(m, 1H), 1.97 (m, 1H), 1.75 (m, 1H), 1.62 (s, 6H).

EXAMPLE 51 (2S,3S)-3-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylicacid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide]

The title compound was prepared using synthetic methodology describedfor the preparation of Example 39 and using(2S,3S)-3-dimethylaminomethyl-pyrrolidine-2-carboxylic acid amide (Stage43.1) instead of (2S,3R)-3-methyl-pyrrolidine-2-carboxylic acid amide inthe last step of the synthesis.

LC-MS: t_(R)=0.91 min, M+H=463, M−H=461 (method L). ¹H-NMR (d₆-DMSO, 400MHz): 10.7 (bs, 1H), 7.36 (s, 1H), 7.01 (bs, 1H), 4.28 (m, 1H), 3.67(dd, 1H), 3.39 (m, 1H), 3.07 (m, 2H), 2.92 (m, 2H), 2.5-2.44 (m, 2H),2.30 (m, 1H), 2.14 (s, 6H), 1.95 (m, 1H), 1.40 (m, 1H), 1.36 (s, 9H).

Efficiency as PI3 Kinase Inhibitors

PI3K KinaseGlo assay: 50 nL of compound dilutions were dispensed ontoblack 384-well low volume Non Binding Styrene (NBS) plates (Costar Cat.No. NBS #3676). L-a-phosphatidylinositol (PI), provided as 10 mg/mlsolution in methanol, was transferred into a glass tube and dried undernitrogen beam. It was then resuspended in 3% OctylGlucoside (OG) byvortexing and stored at 4° C. The KinaseGlo Luminescent Kinase Assay(Promega, Madison, Wis. USA) is a homogeneous HTS method of measuringkinase activity by quantifying the amount of ATP remaining in solutionfollowing a kinase reaction.

5 μL of a mix of PI/OG with the PI3K subtype were added (Table 1).Kinase reactions were started by addition of 5 μl of ATP-mix containingin a final volume 10 μL 10 mM TRIS-HCl pH 7.5, 3 mM MgCl₂, 50 mM NaCl,0.05% CHAPS, 1 mM DTT and 1 μM ATP, and occurred at room temperature.Reactions were stopped with 10 μl of KinaseGlo and plates were read 10mins later in a Synergy2 reader using an integration time of 0.1 secondsper well. 2.5 μM of a pan-class 1 PI3 kinase inhibitor (standard) wasadded to the assay plates to generate the 100% inhibition of the kinasereaction, and the 0% inhibition was given by the solvent vehicle (90%DMSO in water). The standard was used as a reference compound andincluded in all assay plates in the form of 16 dilution points induplicate.

TABLE 1 PI3Ks by KinaseGlo: assay conditions and reagent protocol EnzymeATP PI/OG NaCl Mg²⁺ DTT time Vol (10 μL) (nM) (μM) (μM/μg/ml) (mM) (mM)CHAPS (%) (mM) (mins) PI3Ka 10 1 11/10 50 3 0.05 1 30 PI3Kβ 25 1 11/1050 3 0.05 1 30 PI3Kγ 150 1 22/20 50 3 0.05 1 90 PI3Kd 10 1 11/10 50 30.05 1 30

Cloning of PI3Ks

The PI3Kα, PI3Kβ and PI3Kδ constructs are fusion of p85α iSH2 domain andthe respective p110 isoforms. The p85α fragment and p110 isoform geneswere generated by PCR from first strand cDNA generated by RT-PCR fromcommercial RNA from placenta, testis and brain as described below. ThePI3Kγ construct was obtained from Roger Williams lab, MRC Laboratory ofMolecular Biology, Cambridge, UK (November, 2003) and is described(Pacold, Michael E.; Suire, Sabine; Perisic, Olga; Lara-Gonzalez,Samuel; Davis, Colin T.; Walker, Edward H.; Hawkins, Phillip T.;Stephens, Len; Eccleston, John F.; Williams, Roger L. Crystal structureand functional analysis of Ras binding to its effector phosphoinositide3-kinase gamma. Cell (2000), 103(6), 931-943). PI3Kα Constructs andProteins

PI3Kα wt BV1075 p85iSH2(461-568)-GGGGGGGGGGGG- p110α(21-1068)-His

BV1075: The construct for Baculovirus BV-1075 was generated by athree-part ligation comprised of a p85 fragment and a p110α fragmentcloned into vector pBlueBac4.5. The p85 fragment was derived fromplasmid p1661-2 digested with Nhe/Spe. The p110α fragment derived fromis clone was verified by sequencing and used in a LR410 as aSpeI/HindIII fragment. For the generation of the baculovirus expressionvector LR410 the gateway LR reaction to transfer the insert into theGateway adapted pBlueBac4.5 (Invitrogen) vector was used. The cloningvector pBlueBac4.5 (Invitrogen) was digested with Nhe/HindIII. Thisresulted in the construct PED 153.8. The p85 component (iSH2) wasgenerated by PCR using ORF 318 (described above) as a template and oneforward primer KAC1028 (5′-GCTAGCATGCGAGAATATGATAGAT-TATATGAAG-AATATACC)(SEQ ID No. 1) and two reverse primers, KAC1029(5′-GCCTCCACCAC-CTCCGCCTG-GTTTAATGCTGTTCATACGTTTGTC) (SEQ ID No. 2) andKAC1039 (5′-TACTAGTC-CGCCTCCAC-CACCTCCGCCTCCACCACCTCCGCC) (SEQ ID No.3). The two reverse primers overlap and incorporate the 12× Gly linkerand the N-terminal sequence of the p110α gene to the SpeI site. The 12×Gly linker replaces the single Gly linker in the BV1052 construct. ThePCR fragment was cloned into pCR2.1 TOPO (Invitrogen). Of the resultingclones, p1661-2 was determined to be correct by sequencing. This plasmidwas digested with Nhe and SpeI and the resulting fragment wasgel-isolated and purified for sub-cloning.

The p110α cloning fragment was generated by enzymatic digest of cloneLR410 (see above) with Spe I and HindIII. The SpeI site is in the codingregion of the p110

gene. The resulting fragment was gel-isolated and purified forsub-cloning. The cloning vector, pBlueBac4.5 (Invitrogen) was preparedby enzymatic digestion with Nhe and HindIII. The cut vector was purifiedwith Qiagen column and then dephosphorylated with Calf Intestinealkaline phosphatase (CIP) (BioLabs). After completion of the CIPreaction the cut vector was again column purified to generate the finalvector. A three-part ligation was performed using Roche Rapid ligase andthe vendor specifications. The final plasmid was verified by sequencing.

Kinase domain.

Protein Sequence of BV 1075:

(SEQ ID No. 4) 1 MREYDRLYEE YTRTSQEIQM KRTAIEAFNE TIKIFEEQCQ TQERYSKEYIEKFKREGNEK 61 EIQRIMHNYD KLKSRISEII DSRRRLEEDL KKQAAEYREI DKRMNSIKPGGGGGGGGGGG 121 GLVECLLPNG MIVTLECLRE ATLITIKHEL FKEARKYPLH QLLQDESSYIFVSVTQEAER 181 EEFFDETRRL CDLRLFQPFL KVIEPVGNRE EKILNREIGF AIGMPVCEFDMVKDPEVQDF 241 RRNILNVCKE AVDLRDLNSP HSRAMYVYPP NVESSPELPK HIYNKLDKGQIIVVIWVIVS 301 PNNDKQKYTL KINHDCVPEQ VIAEAIRKKT RSMLLSSEQL KLCVLEYQGKYILKVCGCDE 361 YFLEKYPLSQ YKYIRSCIML GRMPNLMLMA KESLYSQLPM DCFTMPSYSRRISTATPYMN 421 GETSTKSLWV INSALRIKIL CATYVNVNIR DIDKIYVRTG IYHGGEPLCDNVNTQRVPCS 481 NPRWNEWLNY DIYIPDLPRA ARLCLSICSV KGRKGAKEEH CPLAWGNINLFDYTDTLVSG 541 KMALNLWPVP HGLEDLLNPI GVTGSNPNKE TPCLELEFDW FSSVVKFPDMSVIEEHANWS 601 VSREAGFSYS HAGLSNRLAR DNELRENDKE QLKAISTRDP LSEITEQEKDFLWSHRHYCV 661 TIPEILPKLL LSVKWNSRDE VAQMYCLVKD WPPIKPEQAM ELLDCNYPDPMVRGFAVRCL 721 EKYLTDDKLS QYLIQLVQVL KYEQYLDNLL VRFLLKKALT NQRIGHFFFWHLKSEMHNKT 781 VSQRFGLLLE SYCRACGMYL KHLNRQVEAM EKLINLTDIL KQEKKDETQKVQMKFLVEQM 841 RRPDFMDALQ GFLSPLNPAH QLGNLRLEEC RIMSSAKRPL WLNWENPDIMSELLFQNNEI 901 IFKNGDDLRQ DMLTLQIIRI MENIWQNQGL DLRMLPYGCL SIGDCVGLIEVVRNSHTIMQ 961 IQCKGGLKGA LQFNSHTLHQ WLKDKNKGEI YDAAIDLFTR SCAGYCVATFILGIGDRHNS 1021 NIMVKDDGQL FHIDFGHFLD HKKKKFGYKR ERVPFVLTQD FLIVISKGAQECTKTREFER 1081 FQEMCYKAYL AIRQHANLFI NLFSMMLGSG MPELQSFDDI AYIRKTLALDKTEQEALEYF 1141 MKQMNDAHHG GWTTKMDWIF HTIKQHALNE LGGAHHHHHH

PI3Kβ Constructs and Proteins

PI3Kβ BV949 p85iSH2(461-N58K-568)-GGGGGG- p110β(2-1070)-His

BV949: PCR products for the inter SH2 domain (iSH2) of the p85 PI3Kα,PI3Kβ and PI3Kδ subunit and for the full-length p110β subunit weregenerated and fused by overlapping PCR. The iSH2 PCR product wasobtained from first strand cDNA generated by RT-PCR from commercialhuman RNA from placenta, testis and brain (Clontech), initially usingprimers gwG130-p01 (5′-CGAGAATATGATAGATTATATGAAGAAT-3′) (SEQ ID No. 5)and gwG130-p02 (5′-TGGTTT-AATGCTGTTCATACGTTTGTCAAT-3′) (SEQ ID No. 6).Subsequently, in a secondary PCR reaction Gateway recombination AttB1sites and linker sequences were added at the 5′end and 3′end of the p85iSH2 fragment respectively, using primers gwG130-p03(5′-GGGACAAGTT-TGTACAAAAAAGCAGGCTACGAAGGAGATATACATATGCGAGAATATGATAGATTATATGAAGAAT-3′) (SEQ ID No. 7) and gwG130-p05(5′-ACTGAAGCATCCTCCTC-CTCCTCCT-CCTGGTTTAATGCTGTTCATACGTTTGTC-3′) (SEQ IDNo. 8). The p110β fragment was obtained by PCR using as template a p110βclone (from unknown source that was sequence verified) using primersgwG130-p04(5′-ATTAAACCAGGAGGAGGAGGAGGAGGATGCTT-CAGTTTCATAATGCCTCCTGCT-3′) (SEQ IDNo. 9) which contains linker sequences and the 5′end of p110β andgwG130-p06(5′-AGCTCCGTGATGGTGATGGTGATGTGCTCCAGATC-TGTAGTCTTTCCGAA-CTGTGTG-3) (SEQID No. 10) which contains sequences of the 3′end of p110-β fused to aHistidine tag. The p85-iSH2/p110β fusion protein was assembled by anoverlapping PCR a reaction of the linkers at the 3′end of the iSH2fragment and the 5′end of the p110β fragment, using the above mentionedgwG130-p03 primer and a primer containing an overlapping Histidine tagand the AttB2 recombination sequences(5′-GGGACCACTTTGTACAAGAAAGCTGGGTTTAAGCTCCGTGATGGTGATGGTGA TGTGCTCC-3′)(SEQ ID No. 11). This final product was recombined in a Gateway(Invitrogen) OR reaction into the donor vector pDON R201 (Invitrogen) togenerate the ORF253 entry clone. This clone was verified by sequencingand used in a Gateway LR reaction (Invitrogen) to transfer the insertinto the Gateway adapted pBlueBac4.5 (Invitrogen) vector for generationof the baculovirus expression vector LR280. This LR280 has an amino acidmutation in the p85 sequence.

Kinase domain.

Protein Sequence of BV949:

(SEQ ID No. 12) 1 MREYDRLYEE YTRTSQEIQM KRTAIEAFNE TIKIFEEQCQ TQERYSKEYIEKFKREGKEK 61 EIQRIMHNYD KLKSRISEII DSRRRLEEDL KKQAAEYREI DKRMNSIKPGGGGGGCFSFI 121 MPPAMADILD IWAVDSQIAS DGSIPVDFLL PTGIYIQLEV PREATISYIKQMLWKQVHNY 181 PMFNLLMDID SYMFACVNQT AVYEELEDET RRLCDVRPFL PVLKLVTRSCDPGEKLDSKI 241 GVLIGKGLHE FDSLKDPEVN EFRRKMRKFS EEKILSLVGL SWMDWLKQTYPPEHEPSIPE 301 NLEDKLYGGK LIVAVHFENC QDVFSFQVSP NMNPIKVNEL AIQKRLTIHGKEDEVSPYDY 361 VLQVSGRVEY VFGDHPLIQF QYIRNCVMNR ALPHFILVEC CKIKKMYEQEMIAIEAAINR 421 NSSNLPLPLP PKKTRIISHV WENNNPFQIV LVKGNKLNTE ETVKVHVRAGLFHGTELLCK 481 TIVSSEVSGK NDHIWNEPLE FDINICDLPR MARLCFAVYA VLDKVKTKKSTKTINPSKYQ 541 TIRKAGKVHY PVAWVNTMVF DFKGQLRTGD IILHSWSSFP DELEEMLNPMGTVQTNPYTE 601 NATALHVKFP ENKKQPYYYP PFDKIIEKAA EIASSDSANV SSRGGKKFLPVLKEILDRDP 661 LSQLCENEMD LIWTLRQDCR EIFPQSLPKL LLSIKWNKLE DVAQLQALLQIWPKLPPREA 721 LELLDFNYPD QYVREYAVGC LRQMSDEELS QYLLQLVQVL KYEPFLDCALSRFLLERALG 781 NRRIGQFLFW HLRSEVHIPA VSVQFGVILE AYCRGSVGHM KVLSKQVEALNKLKTLNSLI 841 KLNAVKLNRA KGKEAMHTCL KQSAYREALS DLQSPLNPCV ILSELYVEKCKYMDSKMKPL 901 WLVYNNKVFG EDSVGVIFKN GDDLRQDMLT LQMLRLMDLL WKEAGLDLRMLPYGCLATGD 961 RSGLIEVVST SETIADIQLN SSNVAAAAAF NKDALLNWLK EYNSGDDLDRAIEEFTLSCA 1021 GYCVASYVLG IGDRHSDNIM VKKTGQLFHI DFGHILGNFK SKFGIKRERVPFILTYDFIH 1081 VIQQGKTGNT EKFGRFRQCC EDAYLILRRH GNLFITLFAL MLTAGLPELTSVKDIQYLKD 1141 SLALGKSEEE ALKQFKQKFD EALRESWTTK VNWMAHTVRK DYRSGAHHHHHHGA

Kinase domain.

PI3Kγ Construct and Protein

PI3Kγ BV950 p110γ(Δ143-[Met144-1102])-His

Construct obtained from Roger Williams lab, MRC Laboratory of MolecularBiology, Cambridge, UK (November, 2003). Description of the construct in(Pacold, Michael E.; Suire, Sabine; Perisic, Olga; Lara-Gonzalez,Samuel; Davis, Colin T.; Walker, Edward H.; Hawkins, Phillip T.;Stephens, Len; Eccleston, John F.; Williams, Roger L. Crystal structureand functional analysis of Ras binding to its effector phosphoinositide3-kinase gamma. Cell (2000), 103(6), 931-943). Constructs lacking theN-terminal 144 aa. Protein sequence of BV950:

(SEQ ID No. 13) 1 MSEESQAFQR QLTALIGYDV TDVSNVHDDE LEFTRRGLVT PRMAEVASRDPKLYAMHPWV 61 TSKPLPEYLW KKIANNCIFI VIHRSTTSQT IKVSPDDTPG AILQSFFTKMAKKKSLMDIP 121 ESQSEQDFVL RVCGRDEYLV GETPIKNFQW VRHCLKNGEE IHVVLDTPPDPALDEVRKEE 181 WPLVDDCTGV TGYHEQLTIH GKDHESVFTV SLWDCDRKFR VKIRGIDIPVLPRNTDLTVF 241 VEANIQHGQQ VLCQRRTSPK PFTEEVLWNV WLEFSIKIKD LPKGALLNLQIYCGKAPALS 301 SKASAESPSS ESKGKVRLLY YVNLLLIDHR FLLRRGEYVL HMWQISGKGEDQGSFNADKL 361 TSATNPDKEN SMSISILLDN YCHPIALPKH QPTPDPEGDR VRAEMPNQLRKQLEAIIATD 421 PLNPLTAEDK ELLWHFRYES LKHPKAYPKL FSSVKWGQQE IVAKTYQLLARREVWDQSAL 481 DVGLTMQLLD CNFSDENVRA IAVQKLESLE DDDVLHYLLQ LVQAVKFEPYHDSALARFLL 541 KRGLRNKRIG HFLFWFLRSE IAQSRHYQQR FAVILEAYLR GCGTAMLHDFTQQVQVIEML 601 QKVTLDIKSL SAEKYDVSSQ VISQLKQKLE NLQNSQLPES FRVPYDPGLKAGALAIEKCK 661 VMASKKKPLW LEFKCADPTA LSNETIGIIF KHGDDLRQDM LILQILRIMESIWETESLDL 721 CLLPYGCIST GDKIGMIEIV KDATTIAKIQ QSTVGNTGAF KDEVLNHWLKEKSPTEEKFQ 781 AAVERFVYSC AGYCVATFVL GIGDRHNDNI MITETGNLFH IDFGHILGNYKSFLGINKER 841 VPFVLTPDFL FVMGTSGKKT SPHFQKFQDI CVKAYLALRH HTNLLIILFSMMLMTGMPQL 901 TSKEDIEYIR DALTVGKNEE DAKKYFLDQI EVCRDKGWTV QFNWFLHLVLGIKQGEKHSA 961 HHHHHH

PI3Kδ Construct and Protein

PI3Kδ BV1060 p85iSH2(461-568)-GGGGGG- p110δ(2-1044)-His

BV1060: PCR products for the inter SH2 domain (iSH2) of the p85 subunitand for the full-length p110δ subunit were generated and fused byoverlapping PCR. The iSH2 PCR product was generated by using as atemplate the ORF318 (see above) and the primers gwG130-p03(5′-GGGACAAG-TTTGTACAAAAAAGCAGGCTACGAAGGAGATATACATATGC-GAGAATATGATAGATTATATGAAGAAT-3′)(SEQ ID No. 7) and gwG154-p04(5′-TCCTCCTCCT-CCTCCTCCTGGTTTAATGCTGTTCATACGTTTGTC-3′) (SEQ ID No. 14).The p110δ fragment was obtained from first strand cDNA generated byRT-PCR from commercial human RNA from placenta, testis and brain(Clontech), using initially primers gwG154-p01(5′-ATGCCCCCTGGGGTGGACTGCCCCAT-3′) (SEQ ID No. 15) and gwG154-p02(5′-CTACTGCCTGT-TGTCTTTGGACACGT-3′) (SEQ ID No. 16). In a subsequent PCRreaction linker sequences and a Histidine tag was added at the 5′end and3′end of the p110δ fragment respectively, using primers gw154-p03(5′-ATTAAACCAGGAGGAGGAGGAGGAGGACCCCCTGGGGTGGAC-TGCCCCATGGA-3′) (SEQ IDNo. 17) and gwG154-p06(5′-AGCTCCGTGATGGTGATGGTGAT-GTGCT-CCCTGCCTGTTGTCTTTGGACACGTTGT-3′) (SEQID No. 18). The p85-iSH2/p110δ fusion protein was assembled in a thirdPCR reaction by the overlapping linkers at the 3′end of the iSH2fragment and the 5′end of the p110δ fragment, using the above mentionedgwG130-p03 primer and a primer containing an overlapping Histidine tagand the Gateway (Invitrogen) AttB2 recombination sequences(5′-GGG-ACCACTTTGTACAAGAAAGCTGGGTTTAA-GCTCCGTGATGGTGATGGTGAGTGCTCC-3′)(SEQ ID No. 19). This final product was recombined in a Gateway ORreaction into the donor vector pDONR201 (Invitrogen) to generate theORF319 entry clone. This clone was verified by sequencing and used in aGateway LR reaction (Invitrogen) to transfer the insert into the Gatewayadapted pBlueBac4.5 (Invitrogen) vector for generation of thebaculovirus expression vector LR415.

Protein Sequence of BV1060:

(SEQ ID No. 20) 1 MREYDRLYEE YTRTSQEIQM KRTAIEAFNE TIKIFEEQCQ TQERYSKEYIEKFKREGNEK 61 EIQRIMHNYD KLKSRISEII DSRRRLEEDL KKQAAEYREI DKRMNSIKPGGGGGGPPGVD 121 CPMEFWTKEE NQSVVVDFLL PTGVYLNFPV SRNANLSTIK QLLWHRAQYEPLFHMLSGPE 181 AYVFTCINQT AEQQELEDEQ RRLCDVQPFL PVLRLVAREG DRVKKLINSQISLLIGKGLH 241 EFDSLCDPEV NDFRAKMCQF CEEAAARRQQ LGWEAWLQYS FPLQLEPSAQTWGPGTLRLP 301 NRALLVNVKF EGSEESFTFQ VSTKDVPLAL MACALRKKAT VFRQPLVEQPEDYTLQVNGR 361 HEYLYGSYPL CQFQYICSCL HSGLTPHLTM VHSSSILAMR DEQSNPAPQVQKPRAKPPPI 421 PAKKPSSVSL WSLEQPFRIE LIQGSKVNAD ERMKLVVQAG LFHGNEMLCKTVSSSEVSVC 481 SEPVWKQRLE FDINICDLPR MARLCFALYA VIEKAKKARS TKKKSKKADCPIAWANLMLF 541 DYKDQLKTGE RCLYMWPSVP DEKGELLNPT GTVRSNPNTD SAAALLICLPEVAPHPVYYP 601 ALEKILELGR HSECVHVTEE EQLQLREILE RRGSGELYEH EKDLVWKLRHEVQEHFPEAL 661 ARLLLVTKWN KHEDVAQMLY LLCSWPELPV LSALELLDFS FPDCHVGSFAIKSLRKLTDD 721 ELFQYLLQLV QVLKYESYLD CELTKFLLDR ALANRKIGHF LFWHLRSEMHVPSVALRFGL 781 ILEAYCRGST HHMKVLMKQG EALSKLKALN DFVKLSSQKT PKPQTKELMHLCMRQEAYLE 841 ALSHLQSPLD PSTLLAEVCV FQCTFMDSKM KPLWIMYSNE EAGSGGSVGIIFKNGDDLRQ 901 DMLTLQMIQL MDVLWKQEGL DLRMTPYGCL PTGDRTGLIE VVLRSDTIANIQLNKSNMAA 961 TAAFNKDALL NWLKSKNPGE ALDRAIEEFT LSCAGYCVAT YVLGIGDRHSDNIMIRESGQ 1021 LFHIDFGHFL GNFKTKFGIN RERVPFILTY DFVHVIQQGK TNNSEKFERFRGYCERAYTI 1081 LRRHGLLFLH LFALMRAAGL PELSCSKDIQ YLKDSLALGK TEEEALKHFRVKFNEALRES 1141 WKTKVNWLAH NVSKDNRQEL GGAHHHHHH

Purification of PI3Kα, PI3Kβ and PI3Kγ Constructs

PI3Kα, PI3Kβ and PI3Kγ were purified in two chromatographic steps:immobilized metal affinity chromatography (IMAC) on a Ni sepharose resin(GE Healthcare) and gel filtration utilizing a Superdex 200 26/60 column(GE Healthcare). All buffers were chilled to 4° C. and lysis wasperformed chilled on ice. Column fractionation was performed at roomtemperature. All buffers used to purify PI3Kβ contained 0.05% TritonX100 in addition to what is described below.

Typically frozen cells from 10 L of Tn5 cell culture were resuspended in“Lysis Buffer” 20 mM Tris-Cl, pH 7.5, 500 mM NaCl, 5% glycerol, 5 mMimidazole, 1 mM NaF, 0.1ug/mL okadaic acid (OAA), 5 mM BME, 1× Completeprotease inhibitor cocktail—EDTA-free (20 tablets/1 L buffer, RocheApplied Sciences), benzonase (25 U/mL buffer, EMD Biosciences) at aratio of 1:6 v/v pellet to Lysis Buffer ratio, and mechanically lysed bydouncing 20 strokes using a tight-fitting pestle. The lysate wascentrifuged at 45,000 g for 30 minutes, and the supernatant was loadedonto a pre-equilibrated IMAC column (3 mL resin/100 mL lysate). Thecolumn was washed with 3-5 column volumes of Lysis Buffer, followed by asecond wash of 3-5 column volumes with 20 mM Tris-Cl, pH 7.5, 500 mMNaCl, 5% glycerol, 45 mM imidazole, 1 mM NaF, 0.1 μg/mL OAA, 5 mM BME,1× Complete protease inhibitor cocktail—EDTA-free. Protein was elutedwith 20 mM Tris-Cl, pH 7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole, 1mM NaF, 0.1 μg/mL OAA, 5 mM BME, 1× Complete protease inhibitorcocktail—EDTA-free. Pertinent fractions were analyzed by SDS-PAGE andpooled accordingly. The protein was further purified by gel filtrationon a Superdex 200 26/60 column equilibrated in 20 mM Tris-Cl, pH 7.5,0.5 M NaCl, 5% glycerol, 1 mM NaF, 5 mM DTT, 1× Complete proteaseinhibitor cocktail—EDTA-free. Pertinent fractions were analyzed bySDS-PAGE and pooled accordingly. An equal volume of Dialysis Buffer (20mM Tris-Cl, pH 7.5, 500 mM NaCl, 50% glycerol, 5 mM NaF, 5 mM DTT) wasadded to the pool and than dialyzed against Dialysis Buffer two changes(one change overnight). Protein was stored at −20° C.

Purification of PI3Kδ

PI3Kδ was purified in three chromatographic steps: immobilized metalaffinity chromatography on a Ni Sepharose resin (GE Healthcare), gelfiltration utilizing a Superdex 200 26/60 column (GE Healthcare), andfinally a ion exchange step on a Q-HP column (GE Healthcare). Allbuffers were chilled to 4° C. and lysis was performed chilled on ice.Column fractionation was performed at room temperature.

Typically frozen cells from 10 L of Tn5 cell culture were resuspended in“Lysis Buffer” 20 mM Tris-Cl, pH 7.5, 500 mM NaCl, 5% glycerol, 5 mMimidazole, 1 mM NaF, 0.1 μg/mL okadaic acid (OAA), 5 mM BME, 1× Completeprotease inhibitor cocktail—EDTA-free (20 tablets/1 L buffer, RocheApplied Sciences), benzonase (25 U/mL lysis buffer, EMD Biosciences) ata ratio of 1:10 v/v pellet to Lysis Buffer ratio, and mechanically lysedby douncing 20 strokes using a tight-fitting pestle. The lysate wascentrifuged at 45,000 g for 30 minutes, and the supernatant was loadedonto a pre-equilibrated IMAC column (5 mL resin/100 mL lysate). Thecolumn was washed with 3-5 column volumes of Lysis Buffer, followed by asecond wash of 3-5 column volumes with 20 mM Tris-Cl, pH 7.5, 500 mMNaCl, 5% glycerol, 40 mM imidazole, 1 mM NaF, 0.1 ug/mL OAA, 5 mM BME,1× Complete protease inhibitor cocktail—EDTA-free. Protein was elutedwith 20 mM tris-Cl, pH 7.5, 500 mM NaCl, 5% glycerol, 250 mM imidazole,1 mM NaF, 0.1 μg/mL OAA, 5 mM BME, 1× Complete protease inhibitorcocktail—EDTA-free. Pertinent fractions were analyzed by SDS-PAGE andpooled accordingly. The protein was further purified by gel filtrationon a Superdex 200 equilibrated in 20 mM Tris-Cl, pH 7.5, 500 mM NaCl, 5%glycerol, 1 mM NaF, 0.1 μg/mL OAA, 5 mM DTT, 1× Complete proteaseinhibitor cocktail—EDTA-free. Pertinent fractions were analyzed bySDS-PAGE and pooled accordingly. These fractions were diluted 1:10 v/vpool volume to buffer ratio with “Buffer A” 20 mM tris-Cl, pH 8.2, 5%glycerol, 1 mM NaF, 0.1 μg/mL OAA, 5 mM DTT and loaded onto a preparedQ-HP column. After sample loading is completed we wash with Buffer A and5% “Buffer B” 20 mM tris-Cl, pH 8.2, 1 M NaCl, 5% glycerol, 1 mM NaF,0.1 ug/mL OAA, 5 mM DTT for 3-5 column volumes. We elute the proteinusing a 5%-30% gradient of Buffer B. Typically the protein elutes at˜200 mM NaCl. Pertinent fractions were analyzed by SDS-PAGE and pooledaccordingly. An equal volume of Dialysis Buffer (20 mM tris-Cl, pH 7.5,500 mM NaCl, 50% glycerol, 1 mM NaF, 0.1 μg/mL OAA, 5 mM DTT) was addedto the pool and then dialyzed against Dialysis Buffer two changes (onechange overnight). Protein was stored at −20° C.

The following results were obtained using the above described assays.

PI3Kalpha/IC50 PI3Kbeta/IC50 PI3Kgamma/IC50 PI3Kdelta/IC50 Example no.[umol l-1] [umol l-1] [umol l-1] [umol l-1 ] 1 0.004 1.238 0.067 0.18 20.0055 0.332 0.09 0.03 3 0.045 0.127 1.59 0.74 4 0.0065 0.143 0.176 0.225 0.0055 0.047 0.153 0.046 6 0.016 1.63 0.50 0.47 7 0.0235 3.136 0.2330.051 8 0.0095 0.276 0.180 0.198 9 0.0085 0.087 0.141 0.072 10 0.0180.449 0.282 0.054 11 0.005 0.047 0.129 0.018 12 0.006 0.516 0.230 0.05313 0.009 0.277 0.104 0.118 14 0.004 0.268 0.191 0.016 16 0.0055 0.2970.074 0.060 17 0.008 0.389 0.153 0.236 18 0.0105 0.374 0.155 0.028 190.0055 0.588 0.123 0.077 20 0.0035 0.155 0.080 0.011 21 <0.003    0.5190.237 0.036 22 0.0235 6.905 0.262 1.895 24 0.007 0.135 0.098 0.024 250.0055 0.346 0.122 0.045 26 0.0065 0.328 0.138 0.058 27 0.0045 0.3970.097 0.072 28 0.0155 2.035 0.336 0.331 29 0.008 0.721 1.358 0.892 300.023 2.135 0.119 0.294 31 0.0515 5.825 1.226 1.940 32 0.007 1.092 0.1310.043 33 0.0695 5.079 0.909 0.860 34 0.0125 1.818 0.166 0.156 35 0.0092.908 0.195 0.354 36 0.012 0.51 n.d. 0.04 37 0.004 0.182 n.d. 0.019 380.009 0.866 n.d. 0.044 39 0.013 1.612 0.334 0.265 41 0.024 3.046 n.d.0.496 42 0.008 0.409 n.d. 0.032 43 0.005 0.771 n.d. 0.045 44 0.007 0.073n.d. 0.049 45 0.0065 0.061 n.d. 0.024 46 0.0055 1.148 n.d. 0.157 470.003 6.168 n.d. 1.620 48 0.006 0.920 n.d. 0.019 49 0.004 1.248 n.d.0.101 50 0.009 0.555 n.d. 0.113 51 0.005 3.285 n.d. 0.278

1. A compound of formula I

wherein, A is an unsubstituted or substituted aryl ring or unsubstitutedor substituted heterocyclic ring fused to the rest of the molecule atthe positions indicated by the symbol *; X—Y is (CH₂)_(r) or O(CH₂)_(t)or (CH₂)_(t)O wherein, r is 1, 2 or 3; t is 1 or 2; n is 0, 1 or 2; q is0, 1, 2, 3 or 4; R¹ represents, independently at each occurrence, halo;hydroxy; unsubstituted or substituted aryl; unsubstituted or substitutedamino; unsubstituted C₁-C₇-alkyl; C₁-C₇-alkyl, which is substituted oneor more times by hydroxy, C₁-C₇-alkoxy, unsubstituted or substitutedamino, aryl or heterocyclyl, and wherein aryl may be mono orpoly-substituted by halo; or two R¹ substituents together form analkandiyl to form a cyclic moiety, optionally substituted by hydroxy orhalo, or a salt thereof.
 2. A compound according to claim 1, whereinRing A is substituted by one, two or three R² groups, independentlyselected at each occurrence from, unsubstituted or substitutedC₁-C₇-alkyl; unsubstituted or substituted amino; unsubstituted orsubstituted C₃-C₇-cycloalkyl, or a salt thereof.
 3. A compound accordingto claim 1, wherein R² is selected from unsubstituted C₁-C₇-alkyl;di(C₁-C₇-alkyl)amino; C₁-C₇-alkyl substituted one or more times byC₃-C₇-cycloalkyl or halo; unsubstituted C₃-C₇-cycloalkyl;C₁-C₇-cycloalkyl which is substituted one or more times by halo,(halo-C₁-C₇-alkyl) or C₁-C₇-alkyl, or a salt thereof.
 4. A compoundaccording to claim 1, wherein ring A is an unsubstituted or substituted5- or 6-membered ring containing 1 or 2 heteroatoms selected from N, Sor O, wherein at least one heteroatom is N, or a salt thereof.
 5. Acompound according to claim 1, wherein ring A is selected from anunsubstituted or substituted pyridine ring, unsubstituted or substitutedpyrimidine ring or unsubstituted or substituted thiazole ring, or a saltthereof.
 6. A compound according to claim 1, wherein X—Y represents(CH₂)_(r) or O(CH₂)_(t) wherein r is 2 and t is
 1. 7. A compoundaccording to claim 1, wherein R¹ represents, independently at eachoccurrence, halo; hydroxy; unsubstituted or substituted phenyl;di(C₁-C₇-alkyl)amino; unsubstituted C₁-C₇-alkyl; C₁-C₇-alkyl, which issubstituted one or more times by hydroxy, C₁-C₇-alkoxy,di(C₁-C₇-alkyl)amino, di-(perdeuteroC₁-C₇-alkyl)amino, phenyl,morpholinyl, acetylamino, or N—(C₁-C₇-alkyl)-N-(phenylC₁-C₇-alkyl)amino,and wherein independently each phenyl may be mono or poly-substituted byhalo.
 8. A compound according to claim 1, wherein n is 1 and q is
 1. 9.A compound according to claim 1, selected from:(2S,4R)-4-Dimethylamino-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,3S)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(R)-2-Benzyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(S)-2-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(R)-2-Methoxymethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(R)-2-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];d₆-(R)-2-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(R)-2-Hydroxymethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide](R)-2-Hydroxymethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide](R)-2-{[(3-Fluoro-benzyl)-methyl-amino]-methyl}-pyrrolidine-1,2-dicarboxylicacid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(S)-2-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(S)-Azetidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];5-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,4R)-4-Fluoro-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,4S)-4-Fluoro-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(1S,5R)-2-Aza-bicyclo[3.1.0]hexane-1,2-dicarboxylic acid 1-amide2-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(1S,5R)-2-Aza-bicyclo[3.1.0]hexane-1,2-dicarboxylic acid 1-amide2-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,4S)-4-Dimethylamino-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];5-Phenyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];Azetidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(S)-Azetidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,4S)-4-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,4R)-4-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-diethylamino-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide];(S)-2-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide];(S)-Azetidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide];(2S,4R)-4-Dimethylamino-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide];(S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[7-(2-fluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl]-amide};(S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-cyclopropylmethyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide];(2S,3S)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide];(1S,5R)-2-Aza-bicyclo[3.1.0]hexane-1,2-dicarboxylic acid 1-amide2-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide];(2S,3S)-3-(Acetylamino-methyl)-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,3S)-3-Morpholin-4-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,3R)-3-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide];(2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-methyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-amide];(2S,3S)-3-Morpholin-4-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide];(2S,3R)-3-Methoxymethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,3S)-3-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl)-amide];(2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[8-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl]-amide};(2S,3R)-3-Methoxymethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[8-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-thiazolo[4,5-h]quinazolin-2-yl]-amide};(2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-amide];(2S,3S)-3-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(8-tert-butyl-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl)-amide];(2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[8-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4H-5-oxa-1-thia-3,7-diaza-cyclopenta[a]naphthalen-2-yl]-amide};(2S,3R)-3-Methyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[7-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl]-amide};(2S,3R)-3-Methoxymethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-{[7-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl]-amide};(2S,3S)-3-Dimethylaminomethyl-pyrrolidine-1,2-dicarboxylic acid 2-amide1-[(7-tert-butyl-4,5-dihydro-benzo[1,2-d;3,4-d′]bisthiazol-2-yl)-amide].10. A pharmaceutical composition comprising a compound of formula (I),according to claim 1, or a pharmaceutically acceptable salt thereof, andoptionally a further therapeutic agent, together with a pharmaceuticallyacceptable carrier.
 11. A method of treatment of a disease that respondsto inhibition of a lipid and/or protein kinase, which comprisesadministering a prophylactically or therapeutically effective amount ofa compound of formula (I) according to claim 1, or a pharmaceuticallyacceptable salt thereof, to a warm-blooded animal in need of suchtreatment.
 12. A method of treatment according to claim 11, wherein thedisease is a lipid kinase dependent disease dependent on a Class I PI3K.13. A method of treatment according to claim 11, wherein the disease isa lipid kinase dependent disease dependent on a Class I PI3K selectedfrom the group consisting of PI3Kalpha, PI3Kbeta, PI3Kdelta, PI3Kgamma.14. A method of treatment according to claim 11, wherein the disease isa proliferative disease; a benign or malignant tumor; a cancer selectedfrom sarcoma; lung; bronchus; prostate; breast (including sporadicbreast cancers and sufferers of Cowden disease); pancreas;gastrointestinal cancer; colon; rectum; colon carcinoma; colorectaladenoma; thyroid; liver; intrahepatic bile duct; hepatocellular; adrenalgland; stomach; gastric; glioma; glioblastoma; endometrial; melanoma;kidney; renal pelvis; urinary bladder; uterine corpus; uterine cervix;vagina; ovary; multiple myeloma; esophagus; a leukaemia; acutemyelogenous leukemia; chronic myelogenous leukemia; lymphocyticleukemia; myeloid leukemia; brain; a carcinoma of the brain; oral cavityand pharynx; larynx; small intestine; non-Hodgkin lymphoma; melanoma;villous colon adenoma; a neoplasia; a neoplasia of epithelial character;lymphomas; a mammary carcinoma; basal cell carcinoma; squamous cellcarcinoma; actinic keratosis; tumor diseases, including solid tumors; atumor of the neck or head; polycythemia vera; essential thrombocythemia;and myelofibrosis with myeloid metaplasia.